Directed evolution of enzymes and antibodies

ABSTRACT

The invention relates to methods of selecting proteins, out of large libraries, having desirable characteristics. Exemplified are methods of expressing enzymes and antibodies on the surface of host cells and selecting for desired activities. These methods have the advantage of speed and ease of operation when compared with current methods. They also provide, without additional cloning, a source of significant quantities of the protein of interest.

The present application is a continuation of copending U.S. patentapplication Ser. No. 09/813,444, filed Mar. 20, 2001, which is acontinuation of copending U.S. patent application Ser. No. 09/782,672,filed Feb. 12, 2001, which is a continuation-in-part of U.S. Pat. No.5,866,344 filed May 23, 1995 and issued Feb. 2, 1999, which is acontinuation-in-part of U.S. Ser. No. 08/258,543, filed Jun. 10, 1994 isabandoned, which is a divisional of U.S. Pat. No. 5,348,867 issued Sep.20, 1994. The entire text of each of the above-referenced applicationsand patents are specifically incorporated by reference herein withoutdisclaimer. The government owns rights in the present invention pursuantto grant number BCS-9412502 from the National Science Foundation.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of biochemistry,immunology and molecular biology. More particularly, it concerns the useof rapid selection techniques to identify specific polypeptides havingdesirable characteristics out of large libraries of polypeptides.

2. Description of Related Art

There is considerable interest in the discovery of new enzymes withcatalytic and stability characteristics superior to what can be obtainedfrom the pool of naturally-occurring enzymes in the biological world.For practical purposes, it is desirable to identify proteins that (i)can catalyze reactions (e.g., Diels-Alder condensation) that areimportant for chemical processing but do not occur in the biologicalworld; (ii) exhibit unnatural chemical or stereoselectivity; (iii) cancatalyze reactions in the presence of organic solvents ororganic-aqueous mixtures that are typically used in chemical syntheses;and (iv) have an appreciable half-life at elevated temperatures andunder other conditions of interest. Presently, methods for identifyingthese molecules are not readily available.

The development of enzymes with improved characteristics not only hasimportant scientific ramifications, in terms of aiding the understandingof protein structure-function, but also is of great commercial value. Inaddition, most established pharmaceutical and biotechnology companieshave extensive research efforts in place for enzyme engineering andoptimization.

Antibodies also are of increasing importance in human therapy, assayprocedures and diagnostic methods. Therefore, another area of interestlies in the identification of antibodies with particular bindingfunctions, as well as other activities. However, methods of identifyingantibodies and production of antibodies is often expensive, particularlywhere monoclonal antibodies are required. Hybridoma technology hastraditionally been employed to produce monoclonal antibodies, but thesemethods are time-consuming and result in isolation and production oflimited numbers of specific antibodies. Additionally, relatively smallamounts of antibody are produced; consequently, hybridoma methods havenot been developed for a large number of antibodies. This is unfortunateas the potential repertoire of immunoglobulins produced in an immunizedanimal is quite high, on the order of >10¹⁰, yet hybridoma technology istoo complicated and time consuming to adequately screen and developlarge number of useful antibodies.

Selective pressure, i.e., cell cultivation under conditions that allowgrowth only if a certain enzyme is active, has been used for many yearsto isolate mutants with desirable characteristics. The most successfulapproach is to mutagenize in vitro the gene for a desired enzyme,introduce the mutagenized DNA into cells to create a library and finallyselect for cells that produce active enzyme by growing under restrictiveconditions. In one notable example, Liad et al. (1986) reported theisolation of thermostable variants of the E. coli kanamycin nucleotidyltransferase by introducing the gene in a thermophilic bacillus andselecting for mutants that could grow at a temperature above theinactivation temperature of the thermostable enzyme. More recentlyPalzkill et al. constructed sets of large libraries in which blocks ofseveral residues in β-lactamase were randomized by in vitro techniques.The β-lactamase mutant genes were transformed into E. coli and cellscapable of growing in the presence of β-lactam antibiotics that arenormally poor β-lactamase substrates were isolated (Venkatachalam etal., 1994; Petrosino and Palzkill, 1996).

A variety of techniques including chemical mutagenesis of isolated DNA,gene amplification by error prone PCR™ and oligonucleotide mutagenesishave been employed to generate libraries of mutant genes containing adesired range of nucleotide substitutions. Often multiple rounds ofselection and mutagenesis are employed to select increasingly improvedenzymes. It is desirable to be able to combine the beneficial effects ofmutations that exhibit an additive effect on function. For this purposeStemmer (1994a and 1994b) devised a simple technique of DNA shufflingfor allowing the combination of beneficial mutations in different partsof a gene. DNA shuffling is a powerful technique for the generation ofcombinatorial libraries in turn can allow drastic improvements in enzymefunction. As demonstrated by Stemmer (1994a) DNA shuffling allowed arapid isolation of a β-lactamase variant with a 32,000-fold higheractivity towards the antibiotic cefotaxime.

The selection of improved enzymes from in vitro constructed libraries isa very powerful approach for biocatalyst design provided that the enzymesought confers an essential function for the cell. Unfortunately in manycases of commercial interest it is not possible to design a selectionstrategy. In fact it is impossible to design selection strategies forreactions (such as Diels Alder condensation) that do not take place inbiological systems. The other limitation of mutant selection strategiesis that under selective conditions cells can evolve mechanisms ofsurvival that bypass the reaction catalyzed by the enzyme that is to beoptimized. The ability of cellular adaptation mechanisms to respond toselective pressure has frustrated efforts to direct the evolution ofefficient antibody catalysts designed to complement mutations inessential pathways within microorganisms (Tang et al., 1991; Smiley andBenkovic, 1994).

For enzymatic reactions where the design of a selection strategy is notpossible, libraries of mutants have to be screened by direct assay. Thisinvolves growing individual clones either as colonies on agar plates or,alternatively in 96-well plates, and measuring enzymatic activityusually by a chromogenic assay. A popular and convenient screeningformat is to determine the enzymatic activity of single colonies growingon agar plates. Sequential cycles of random mutagenesis and platescreening are increasingly employed for the directed evolution of enzymeactivities. Using this approach Moore and Arnold (1996) isolated amutant paranitrobenzyl esterase that exhibits 16-fold higher activity in30% DMF relative to the parent enzyme. At least one other enzyme hasbeen engineered successfully by random mutagenesis and screening (Yu andArnold, 1996).

However, the isolation of clones producing desired enzymes byplate-screening is tedious and is not suitable when a drastic change inprotein function or stability is sought. It is impractical to screenmore than 10⁵ clones by plate assays (even with automated techniques)and therefore only a relative small set of mutants can be analyzed.Screening of much larger libraries of mutants, typically comprising ofat least 1,000-fold larger number of clones (i.e., 10⁸) is necessary ifthere is to be any hope for completely changing enzyme activity orreversing its substrate specificity.

The screening of colonies using plate assays suffers form threeadditional limitations. First, plate assays can be devised for a limitedrange of reactions. Second, because the vast majority of proteins arenot released from E. coli bacteria (by far the most preferred hostorganism for “directed evolution” studies), the substrate must be ableto readily diffuse into the cell and it must not be toxic. Third, plateassays, even those that utilize fluorescent molecules have at bestmoderate specificity. In recent years there have been many efforts tofind rapid assay screening methods that can circumvent the limitationsinherent with plate assays. Perhaps the most innovative approach is theCatalytic ELISA (CatELISA) technique developed by Tawfik et al. (1993).However, neither CatELISA nor any of the other assay recent techniquescan be applied to the screening of larger libraries of mutants.

Thus despite the intense interest in discovering new enzymes andantibodies, there remain a significant technical hurdles that have madeit difficult to exploit this considerable wealth of biological power.Thus, there remains a great need for improved methods of handling thesynthesis and identification of the vast number of possible activepolypeptides.

SUMMARY OF THE INVENTION

The present invention addresses these and other drawbacks inherent inthe prior art by providing new methods of screening of polypeptidelibraries. For the first time it is possible to rapidly screenpolypeptide libraries for potential enzymes and antibodies; often in amatter of hours. The disclosed methods allow production of largequantities of these polypeptides, potentially on a kilogram scale, frommicroorganism cultures. And, because selected proteins can be displayedon the surface of a host cell, assays can be conducted with remarkablerapidity.

In one aspect of the invention, expression libraries are prepared suchthat an expressed protein is displayed on the surface of a cell.Typically, the polypeptides will be surface expressed in a host cellsuch as bacterial, yeast, insect, eukaryotic or mammalian cells. Surfaceexpression of a polypeptide on a cell surface is achieved using arecombinant vector that promotes display on the outer membrane of a hostcell. Vectors are such as those of the general construction described inU.S. Pat. No. 5,348,867, incorporated herein by reference. Generally thevectors will be appropriate for a bacterial host cell and will includeat least three DNA segments as part of a chimeric gene. One segment is aDNA sequence encoding a polypeptide that targets and anchors a fusionpolypeptide to a host cell outer membrane. A second DNA segment encodesa membrane-transversing amino acid sequence, i.e., a polypeptide thattransports a heterologous or homologous polypeptide through the hostcell outer membrane. The third DNA segment encodes any of a number ofdesired polypeptides. Such vectors will display fusion polypeptides atthe exterior of a host cell. These recombinant vectors include afunctional promoter sequence.

Screening for antibodies employing the methods of the present inventionallows one to select an antibody or antibody fragment from a pluralityof candidate antibodies that have been expressed on the surface of ahost cell. In most instances the host cell will be a bacterial cell,preferably E. coli. The antibodies are obtained from an expressionvector library that may be prepared from DNAs encoding antibodies orantibody fragments. One source of such DNAs could be from an animalimmunized with a selected antigen; alternatively, antibody genes fromother sources can be used, such as those produced by hybridomas orproduced by mutagenesis of a known antibody gene. One preferred methodof obtaining DNA segments is to isolate mRNA from antibody cells of animmunized animal. The mRNA may be amplified, for example by PCR, andused to prepare DNA segments to include in the vectors. One may alsoemploy DNA segments that have been mutagenized from one or more DNAsthat encode a selected antibody or antibody fragment.

In a second embodiment, the present invention provides methods for therapid screening of enzyme libraries. The libraries represent mutagenizedversion of an enzyme to permit for the “directed” evolution of theenzyme's sequence, and hence function. Again, vectors will comprise aDNA sequence encoding the enzyme, an anchor fused to the enzyme codingregion that results in expression of the host cell outer membrane andany other regulatory sequences necessary for the propagation of thevectors and the expression of the enzyme. Standard mutagenic procedureswill be applied to various regions of the enzyme coding region,including those regions that encode binding pockets and active sites.Other sites of interest, including residues critical for conformationand post-translational modification also may be targeted.

Once generated, expression libraries are transferred, by standardmethodologies, into appropriate host cells. Expression of theantibodies, enzymes or other polypeptides on the surface of host cellspermits the rapid and efficient screening of libraries for theappropriate binding specificity, enzyme function or other desirablecharacteristic. In addition, use of appropriate label systems andsubstrates permits the sorting of host cells expressing proteins ofinterest by flow cytometery methodology (e.g., FACS).

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

FIG. 1A and FIG. 1B. FIG. IA Western blot analysis of total membranefractions from E. coli JM109 cells containing pTX101 (Lanes 2 and 4);JM109/pTX152 (Lanes 3 and 5) and probed with anti-OmpA antibodies at1:5000 dilution; Lanes 4 and 5 were probed with monoclonal anti-HSVantibodies at 1:5000 dilution. Arrowheads indicate theLpp-OmpA-β-lactamase fusion (lane 2) and the Lpp-OmpA-scFv(digoxin)fusion (lane 3). The 32 kDa band in lanes 2 and 3 corresponds to OmpA.Lane 1, molecular mass markers (in kDa). FIG. 1B. Lysate and whole cellELISAs of JM109 cells containing plasmid pTX101 (solid) or pTX152(hatched). Samples were incubated on microtiter wells coated withdigoxin-conjugated BSA and probed with anti-β-lactamase (pTX101) oranti-HSV (pTX152) antibodies. Absorbance readings were referenced towells that were untreated with either lysates or whole cells.

FIG. 2A and FIG. 2B. Phase contrast micrograph of JM109/pTX152 cellsafter 1 hr of incubation with 10⁻⁷ M digoxin-FITC. FIG. 2B. Micrographsof the same field as in FIG. 2A of JM109/pTX152 cells after a 1 hourincubation with 10⁻⁷M digoxin-FITC.

FIGS. 3A-3G. Histogram data from FACS. The bar in each graph representsthe sorting gate or the fluorescence intensity defined as a positiveevent. The sorting gate was chosen to maximize the number of positiveevents while minimizing the number of negative events within the window.All samples were labeled with 10⁻⁷ M digoxin FITC. FIG. 3A. JM109/pTX152sample used as a negative control. FIG. 3B. JM109/pTX152 sample used asa positive control. FIG. 3C. JM109/pTX152 pretreated with 0.2 mg/mltrypsin. FIG. 3D. JM109/pTX152 pretreated with free digoxin. FIG. 3E. A100,000:1 mixture of JM109/pTX101:JM109/pTX152 prior to the first cellsorting run. FIG. 3F. A 100,000:1 mixture after growing cells recoveredfrom the first cell sorting run. FIG. 3G. A 100,000:1 mixture aftergrowing cells recovered from the second cell sorting run.

FIG. 4. Whole cell immunoassay using 0.5 nM FITC labeled digoxin.

FIG. 5A and 5B: FIG. 5A. Antibody mutants displaying different affinityfor the antigen can be distinguished by display on the cell surface andfluorescence activated cell sorting. A. Fluorescence histogram comparingthe fluorescence distribution of bacterial cells displaying mutants ofthe svFv (digoxin) antibody on their surface. FIG. 5B. Relative bindingaffinity of the corresponding purified antibodies measured by ELISA.

FIG. 6A and 6B Immunoassay for the determination of the equilibriumconstant for antigen binding for surface displayed scFv antibodies.Cells displaying scFv(digoxin) antibodies on their surface wereincubated with different concentrations of BODIPY-digoxin for one hourwith gentle shaking. Following incubation, the fluorescence distributionof the cells (50,000 events) was determined by flow cytometry.

FIG. 7: Single chain antibody surface display plasmid vector pSD192

FIG. 8: Procedure for the isolation of single-chain antibodies bysurface display and FACS.

FIG. 9A-9F: Isolation of high affinity antibodies from librariesdisplayed on the bacterial cell surface and screened by FACS. FIG.9A-9C: Plots of Forward Scatter (a measure of the cell size) as afunction of fluorescence intensity for cell populations displaying alibrary of scFv antibodies and incubated with different concentrationsof fluorescent hapten (BODIPY-digoxin). The library was constructed byrandomizing the heavy chain residues 99, 100, 100a and 100b as describedin Example 7. Cells were incubated with Bodipy-digoxin at differentconcentrations, shown in FIG. 9A-9C, for one hour. Each point representsone event detected by the flow cytometer and a total of 50,000 events(i.e. cells) are shown for clarity. FIG. 9D-9F. Isolation of scFvantibodies from a library. The library was constructed by randomizingthe heavy chain residues 99, 100, 100a and 100b as described in Example7. Cells were incubated with 70 nM BODIPY-digoxin and high fluorescenceclones were isolated by FACS. The corresponding fluorescence histogramis shown in FIG. 9D. Sorted cells were grown overnight, incubated with15 nM BODIPY-digoxin and sorted. The fluorescence distribution of thecells is shown in FIG. 9E. As can be seen, after one round of sortingand growth, cells having high fluorescence are greatly enriched over thestarting cell population. Finally, FIG. 9F shows the fluorescencedistribution of the cell population obtained after growing the positivecells isolated in the first round. The large majority of the cells bindthe BODIPY-digoxin conjugate and thus have a high fluorescence.

FIG. 10: Chemical Structure of the OmpT substrate

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

A. Identifying Polypeptides With Desirable Properties

As stated above, the natural diversity of biologically activepolypeptides, in terms of both function and specificity, is immense. Theproblem lies in tapping this vast reservoir of potential reagents in asufficiently expedient fashion. In other words, how does one identifythe few polypeptides out of the almost infinite number of possibilitiesthat function as desired? Automated selection technologies may speed upthe process, but the real obstacle remains the sheer number ofpossibilities—how they can be generated and how they can be expressed inorder for the selection to take place. The present invention is designedto address this particular problem, as described in greater detailbelow.

Enzymes

Efforts to engineer improved enzymes rely upon molecular biologytechniques and involve two basic approaches. The first involves cloningand expression of enzyme libraries from organisms that cannot becultivated and typically are isolated from extreme environments. Thisapproach, pioneered by Recombinant Biocatalysis (Brennan, 1996), relieson the construction expression libraries by extracting DNA from samplesand gene amplification by PCR™. The expression libraries are thenscreened by brute force approaches that rely heavily on robotics. Thistechnology promises to begin to tap the unexplored diversity of functionin the natural world. However, it is intrinsically limited to catalyticactivities that serve a biological function. Also, it is limited by theability to employ genes from unknown sources to direct the synthesis offunctional properties in commonly used host organisms such as E. coli orSaccharomyces cerevisiae.

A second approach relies on selecting antibodies with the appropriateenzymatic or catalytic characteristics. The principle underlyingcatalytic antibodies is straightforward—binding of an antibody to itscognizant antigen results in a free energy decrease. An antibody that ishighly complementary to the rate-limiting species of a chemical reaction(i.e., the transition state complex) will lower the energy of thatspecies. This decrease in the free energy of formation of the transitionstate translates into a higher rate of reaction. In general terms,catalytic antibodies are produced via immunization with a molecule (ahapten) that is designed to mimic certain features of the transitionstate complex for the reaction of interest (Lemer et al., 1991). Animpressive array of different reactions now have been catalyzed byantibodies. Moreover, as expected for a catalyst based on antibodyrecognition, catalytic antibodies display precise substrate selectivity.That is, only substrates that are similar in structure to the haptenused to elicit the catalytic antibodies are accepted in the catalyticreaction.

Unfortunately, catalytic antibodies have not yet fulfilled initialexpectations. A number of reasons are at the root of the slowprogression. First, the generation of catalytic antibodies istechnically difficult and prohibitively expensive. Second, productioncosts are uneconomical. Despite impressive advances in hybridoma culturescale-up, the cost-effective production of monoclonal antibodies remainsa serious challenge. Third, poor kinetic properties are common.Typically, rates of reaction and acceleration with catalytic antibodies(i.e., rate of catalyzed reaction over uncatalyzed reaction) are between10⁴-10⁵, although a few examples of higher rates have been reported(Janda et al., 1988; however also see Hollfelder et al., 1996). Forcomparison, rates of reaction acceleration for enzymes are usuallyaround 10⁷-10⁸. Fourth, transition state mimics must be immunogenic andstable in vivo. Animal immunization with the transition state analog isthe first step in the production of monoclonal antibodies with catalyticactivity. The need for immunization poses two serious constraints on thetransition state analog; it must be recognized by the immune responseand it must be stable in the animal.

Antibodies

Currently, the most widely used approach for screening polypeptidelibraries is to display polypeptides on the surface of filamentousbacteriophage (Smith, 1991; Smith, 1992). The polypeptides are expressedas fusions to the N-terminus of a coat protein. As the phage assembles,the fusion proteins are incorporated in the viral coat so that thepolypeptides become displayed on the bacteriophage surface. Eachpolypeptide produced is displayed on the surface of one or more of thebacteriophage particles and subsequently tested for specific ligandinteractions. While this approach appears attractive, there are numerousproblems, including difficulties of enriching positive clones from phagelibraries. Enrichment procedures are based on selective binding andelution onto a solid surface such as an immobilized receptor.Unfortunately, avidity effects arise due to multivalent binding of thephage and the general tendency of phage to contain two or more copies ofthe displayed polypeptide. The binding to the receptor surface thereforedoes not depend solely on the strength of interaction between thereceptor and the displayed polypeptide. This causes difficulties in theidentification of clones with high affinity for the receptor; thus,there remain distinct deficiencies in the methods used to isolate andscreen polypeptides, particularly antibodies, even in view of thedevelopment of phage libraries.

Previous Attempts to Develop Engineered Polypeptides

An approach to the antibody selection problem has been the developmentof library screening methods for the isolation of antibodies (Huse etal, 1989; McCafferty et al., 1990; Chiswell & McCafferty, 1992; Chiswell& Clackson, 1992; Clackson, 1991). Functional antibody fragments havebeen produced in E. coli cells (Skerra & Pluckthun, 1988; Better et al.,1988; Orlandi et al., 1989; Sastry et al., 1989) as “libraries” ofrecombinant immunoglobulins containing both heavy and light variabledomains (Huse et al, 1989). The expressed proteins have antigen-bindingaffinity comparable to the corresponding natural antibodies. However, itis difficult to isolate high binding populations of antibodies from suchlibraries and where bacterial cells are used to express specificantibodies, isolation and purification procedures are usually complexand time-consuming.

Combinatorial antibody libraries generated from phage lambda (Huse etal, 1989) typically include millions of genes of different antibodiesbut require complex procedures to screen the library for a selectedclone. Ladner et al., (U.S. Pat. No. 5,403,484, specificallyincorporated herein by reference) reported the display of proteins onthe outer surface of a chosen bacterial cell, spore or phage, in orderto identify and characterize binding proteins. Certain elements ofLadner may be used advantageously, for example, methods of generatingand expressing single chain antibodies, proteinaceous binding domainsother than a single chain antibody, carrier protein and the like, incombination with the present invention.

Methods have been reported for the production of human antibodies usingthe combinatorial library approach in filamentous bacteriophage. A majordisadvantage of such methods is the need to rely on initial isolation ofthe antibody DNA from peripheral human blood to prepare the library.

One way of approaching enzyme selection relies on the possibility ofobtaining accurate 3-dimensional structures for polypeptides, which thencan be employed to identify amino acid substitutions that can alter orenhance function. Structure-guided mutagenesis in protein chemistry isbest exemplified by the elegant and extensive studies of Matthews andcoworkers using the T4 lysozyme as a model (Matthews, 1995). Over theyears his group constructed, characterized and solved the crystalstructure of over 100 T4 mutants. These studies have provided valuableinformation on the determinants of protein stability. A few mutants withmarkedly increased thermostability have been isolated. However,structure guided mutagenesis is not likely to become a general route tothe isolation of new biocatalysts because, (i) the three dimensionalstructure of a protein is required as a starting point, and the threedimensional structure is not available for most polypeptides and (ii)while the prediction of amino acid substitutions that affect stabilityhas been met with some success, mutations that increase or dramaticallyalter catalytic activity have been very difficult to predict.

B. The Present Invention

It is proposed, according to the present invention, to provide a generalapproach for the efficient screening of very large libraries ofvirtually any polypeptide for desirable activity. This approach employsdirected evolution for the selection of enzymes and, further, can beapplied to the screening of antibody libraries. This provides anextremely powerful tool for the selection of such polypeptides.Moreover, the development of methods that achieve stable anchoring andpolypeptide display on the surface of a bacterial cell such as E. coli,has provided the basis for new methods of rapid and efficient selectionof polypeptides from libraries expressed in the host cells. With thepolypeptide on the surface, exposed directly to the aqueous media, theactivity of the polypeptide can be assayed directly, without having toworry about transport of assay reagents into cells.

The methods disclosed herein are particularly advantageous because theyallow unprecedented rapid and efficient selection, purification andscreening of polypeptide libraries from bacterial host cell surfaces,providing several advantages over phage libraries. Unlike most othermethods used for screening and assay, the disclosed methods arewell-suited for commercial adaptation. Assay procedures are greatlyfacilitated because of the cell surface display aspect, permitting theuse of simple centrifugation to remove the cells from an assay sample.Assays thus are very rapid and inexpensive as they do not requirecomplex or expensive equipment.

In a first embodiment, relating to directed enzyme evolution, theinvention comprises the following approach. A target enzyme (orcatalytic antibody) which is to be subjected to mutagenesis is firstdisplayed on the surface of E. coli bacteria as a fusion to asurface-targeting vehicle. This technology was developed by Georgiou andcoworkers and has been used to display a number of proteins on thebacterial surface (Francisco et al., 1992; 1993a; 1993b; Georgiou etal., 1996). By displaying the target enzyme on the bacterial surface itwill be fully accessible to molecules in the extracellular fluid. Thus,the enzyme is free to react with any substrate added to the cellswithout any of the limitations that are imposed when intracellularenzymes are studied.

Expression of recombinant proteins on cell surfaces of Gram-negativebacteria is achieved by fusion to segments of a major lipoprotein andOmpA; however, fusion to protein domains other than those derived fromthe major lipoprotein and OmpA is also envisioned, provided that thesedomains can function for the expression of the desired polypeptide onthe cell surface. Generally, the desired polypeptide is fused to anamino acid sequence that includes the signals for localization to theouter membrane and for translocation across the outer membrane. Theamino acid sequences responsible for localization and for translocationacross the outer membrane may be derived either from the same bacterialprotein or from different proteins of the same or different bacterialspecies. Examples of proteins that may serve as sources of localizationsignal domains are shown in Table 1. TABLE 1 EXAMPLES OF OUTER MEMBRANETARGETING SEQUENCES Organism Lpp E. coli (or functional equivalent inSalmonella) TraT E. coli (or functional equivalent in Salmonella) OsmBE. coli (or functional equivalent in Salmonella) N1pB E. coli (orfunctional equivalent in Salmonella) BlaZ E. coli (or functionalequivalent in Salmonella) Lpp1 Pseudomonas aeruginosa PA1 Haemophilusinfluenza OprI E. coli 17 kDa lpp Riokettsia riokettsii H.8 proteinNeisseria gonorrhea

In addition a sequence that allows display of the polypeptide on thecell surface is required. Appropriate examples are shown in Table 2.TABLE 2 EXAMPLES OF TRANSMEMBRANE SEQUENCES OmpA E. coli or functionalequivalent in Salmonella LamB E. coli or functional equivalent inSalmonella PhoE E. coli or functional equivalent in Salmonella OmpC E.coli or functional equivalent in Salmonella OmpF E. coli or functionalequivalent in Salmonella OmpT E. coli or functional equivalent inSalmonella FepA E. coli or functional equivalent in Salmonella

Several fusion protein strategies for the display of relatively shortpeptides on the surface of Gram-negative bacteria have been described(Table 3). Short peptides of less than 60 amino acids residues can bedisplayed on the cell surface when fused into surface exposed loops ofouter membrane proteins (OMPs) from enteric bacteria (Hofnung et al.,1991; Charbitt et al., 1988; Agterberg et al. 1990; Su et al., 1992;Wong et al., 1995; Newton et al., 1996). Hofnung and coworkers were thefirst to demonstrate that peptides inserted within permissive sites ofthe E. coli outer membrane protein LamB are displayed on the cellsurface, accessible to antibodies in the extracellular fluid and havethus been exploited extensively for practical applications Hofnung etal., 1991; Brown, 1992; O'Callaghan et al., 1990; Sousa et al., 1996).However, it was quickly realized that the insertion of peptides longerthan 60 amino acids perturbs the overall conformation and assembly ofthe carrier, interfering with the localization of the fusion proteins(Hofnung et al., 1991; Charbitt et al., 1988; Agterberg et al. 1990).Moreover, the positioning and length of the peptide insert plays acritical role in the efficient surface display and recognition of theinserted epitope (Su et al., 1992; Wong et al., 1995; Newton et al.,1996). TABLE 3 Expression Systems for protein display in E. coli CarrierType of Fusion Localization of Passenger Passenger PolypeptideApplications E. coli LamB sandwich fusion cell surface variety of viralpeptide vaccines, peptide libraries, antigen cellular absorbants E. coliPhoE sandwich fusion cell surface epitope from hsp65 of M. vaccinestuberculosis Pseudomonas OprF sandwich fusion cell surface 4 aa epitopefrom malaria vaccines parasite E. coli or other C-terminal orperiplasmic side of outer scFv antibodies; 11 aa CE lipid taggedantibodies, Gram-negative sandwich fusions membrane/cell surface epitopeof polio virus vaccines lipoprotein E. coli Lpp-OmpA C-terminal fusioncell surface scFv antibodies; β- peptide/antibody libraries, lactamase;protein A; cellular adsorbents, cellulose binding protein immunoassaysShigella VirG₈ N-terminal fusion cell surface alkaline phosphataseNeisseria IgA₈ N-terminal fusion cell surface cholera toxin B subunitvaccines peptide libraries E. coli Flagellin sandwich fusion cellsurface thioredoxin; peptides peptide libraries (Flic) inserted withinthioredoxin Salmonella sandwich fusion cell surface 18 aa epitope fromHIV1 vaccines Flagellin (FliC) gp41 protein E. coli FimH (Type sandwichfusion cell surface 52 aa sequence from the vaccines I pili) preS2hepatitis B antigen E. coli PapA sandwich fusion cell surface 58 aadomain from cellular adsorbents Staphylococcus protein A Klebsiella PulAC-terminal fusion cell surface/extracellular fluid B-lactamase

In a different approach, Fiers and colleagues expressed theimmunoglobulin G-binding domain of protein A of Staphylococcus aureus onthe surface of E. coli using PapA, the major subunit of the Pap pilus(Steidler et al., 1993). Other groups have used flagellum or pilussubunits to develop expression systems for the surface presentation ofantigenic/immunogenic epitopes derived from pathogens, suitable for thedevelopment of live recombinant vaccines (Newton et al., 1995; Pallesenet al., 1995; Van Die et al., 1990).

For reasons that are not fully understood, subunits of cellularappendages and outer membrane porins are not suitable for the surfacedisplay of large polypeptides. To overcome this problem it has beennecessary to use surface display carrier proteins that are exported viamore specialized mechanisms (Salmong et al., 1993). For example, thetargeting of many lipoproteins from Gram-negative bacteria onto theouter membrane is determined only by the presence of a short N-terminalsequence. Because of this property, several lipoproteins have beentested as potential carriers for surface display (Taylor et al., 1990;Laukkanen et al., 1993; Fuchs et al., 1991; Cornellis et al., 1996).Unfortunately, lipoprotein fusions have been found to be eitherdetrimental to the integrity of the cell envelope causing extensive celllysis, or to be tethered to the interior face of the outer membrane, inwhich case they are not exposed to the extracellular fluid (Laukkanen etal., 1993;Cornellis et al., 1996).

These limitations have been addressed by constructing an Lpp-OmpA hybriddisplay vehicle consisting of the N-terminal outer membrane localizationsignal from the major lipoprotein (Lpp) fused to a domain from the outermembrane protein OmpA (Franscisco et al., 1992). OmpA mediates thedisplay of passenger proteins fused to the C-terminal of the Lpp-OmpAhybrid. Lpp-OmpA fusions have been used to successfully display on thesurface of E. coli several proteins varying in size between 20 and 54kDa (Stathopoulos et al., 1996). Among the proteins that have beentested thus far only the dimeric bacterial enzyme alkaline phosphatase(phoA) could not be displayed on the cell surface (Stathopoulos et al.,1996).

The IgA proteases of Neisseria gonorrhoeae and Hemophilus influenzae usea variation of the most common, Type II secretion pathway (Salmong etal., 1993), to achieve extracellular export independent of any othergene products (Klauser et al., 1993). Specifically, the C-terminaldomain of the IgA protease forms a channel in the outer membrane thatmediates the export of the N-terminal domain across the membrane whichin turn becomes transiently displayed on the external surface of thebacteria. This export mechanism is used by a number of extracellularproteins from pathogenic bacteria (Klauser et al., 1993; Jose et al.,1995; Suzuki et al., 1995; Provence et al., 1997; St. Geme et al.,1994). Replacement of the native N-terminal domain of IgA protease orVirG with the cholera toxin B subunit or the periplasmic E. colicprotein MalE, respectively, resulted in the surface presentation of thepassenger polypeptides (Suzuki et al., 1995; Klauser et al., 1990).

Unlike the IgA protease, the lipoprotein pullulanase (PulA) ofKlebsiella pneumoniae, which is also exported via a type Ii secretionmechanism, requires 14 genes for its translocation across the outermembrane (Salmong et al., 1993). Pugsley and coworkers have shown thatthe lipoprotein pullulanase (PulA) can facilitate translocation of theperiplasmic enzyme 13-lactamase across the outer membrane. However,pullulanase hybrids remain only temporarily attached to the bacterialsurface and are subsequently released into the medium (Komacker et al.,1990). Although the lack of permanent association with the cell wall isnot detrimental for vaccine development, it is a serious limitation inother applications such as library screening.

Expression systems for the display of proteins in Gram-positive bacteriahave also been developed (Fischetti, 1996). Uhlen and colleagues usedfusions to the cell-wall bound, X-domain of protein A, for the displayof foreign peptides up to 88 amino acids long to the surface ofStaphylococcus strains (Hansson et al., 1992; Samuelson et al., 1995).In other studies, the fibrillar M6 protein of Streptococcus pyogenes wasemployed as a carrier for antigen deliver in Streptococcus cells (Pozziet al., 1992).

Protein display applications has also spurred the development ofsuitable expression systems for yeast cells. Surface display expressionsystems for yeast have relied primarily on the fusion of passengerproteins to agglutinin, a protein involved in cell adhesion (Schreuderet al., 1993; Schreuder et al., 1996; Schreuder et al., 1996; Boder andWittrup et al 1996). The AGα1 agglutinin is tightly bound to the cellwall through its C-terminus. N-terminal fusions to the cell wall domainof AGA1 are stably anchored on the cell surface. This system has beenused for the surface expression of a variety of enzymes and bindingproteins (Schreuder et al., 1996). Mating-type a cells use the twosubunit agglutinin a for cell adhesion. Recently the second subunit ofagglutinin a (Aga2p) was used as a vehicle for the surface display ofantibodies and peptides (Boder and Wittrup et al., 1996). In this case,the passenger polypeptide is fused to the C-terminus of AGA2 which, inturn, is linked to the AGA1 via disulfide bonds.

The gene encoding the target enzyme is mutagenized by conventionaltechniques to generate a library of mutants. The library of mutants willbe screened using a highly sensitive single cell assay. Cells exhibitingthe desired activity will be isolated. This will involve the following:(i) design of a fluorescent substrate for the desired reaction; and mayinvolve immobilization of the cells onto micron-size particles; and (ii)screening and isolation of fluorescent microparticles either byfluorescent activated cell sorting or by using a micromanipulator.

In a second embodiment, the present invention relies on the same cellsurface display of polypeptides as described above, except that thepolypeptide to be expressed on the host cell surface is an antibody. Thescreening methods are practiced by first constructing an antibodylibrary, using any of several well-known techniques for libraryconstruction. For example, after selecting an immunogen, one mayimmunize a mammal by conventional means and collect antiserum. mRNA fromspleen may be used as template for PCR amplification; for exampleemploying primers complementary to constant and variable domainframework regions of different antibody subclasses. Alternatively, DNAfrom polyclonal populations of antibodies may be amplified, fragmentedif desired, ligated into pTX101 or a similar vector as described in U.S.Pat. No. 5,348,867, incorporated herein by reference, and transformedinto a host cell.

C. Expression Libraries and Mutagenesis

The present invention involves the display of polypeptides on thesurface of a bacteria. U.S. Pat. No. 5,348,867, addressing this topic,is specifically incorporated by reference. As used herein, the termpolypeptide refers to any protein and includes antibodies, antibodyfragments, receptors, enzymes, cytokines, transcription factors,clotting factors, chelating agents and hormones.

Genes for polypeptides of interest are fused to the 3′ of a sequencethat encodes a cell surface targeting domain. The cell envelope of E.coli and other gram-negative bacteria consists of the inner membrane(cytoplasmic membrane), the peptidoglycan cell wall and the outermembrane. Although the latter normally serves as a barrier to proteinsecretion, a targeting sequence has been developed that, when fused tonormally soluble proteins, can direct them to the cell surface(Francisco et al., 1992; 1993). The surface targeting domain includesthe first nine amino acids of Lpp, the major lipoprotein of E. colifused to amino acid 46-159 of the Outer Membrane Protein A (OmpA). Thefunction of the former is to direct the chimera to the outer membranewhereas the OmpA sequence mediates the display of proteins at theC-terminal of OmpA. Lpp-OmpA(46-159) fusions have been used to anchor avariety of proteins such as β-lactamase, a cellulose binding protein andalkaline phosphatase on the E. coli surface. However, other analogoussurface targeting domains may be employed to stably anchor therecombinant polypeptides on the cell surface.

Methods for the display of virtually any polypeptide on the surface E.coli are described in U.S. Pat. No. 5,348,867. Any library encoding aset of related polypeptide sequences may be displayed on the surface ofE. coli. Examples of such libraries include, libraries derived bymutagenesis of a homologous or heterologous protein, random peptidelibraries, epitope libraries and libraries of recombinant antibodyfragments, all of which may be created by methods known to those skilledin the art.

Exemplary of the surface expression method is an Lpp-OmpA(46-159)-antibody fusion expressed in a gram-negative bacterium. Due tothe presence of the Lpp-OmpA(46-159) sequence, the fusion is localizedon the outer membrane such that the N-terminal domain is embedded in thebilayer and the antibody sequence is fully exposed on the cell surface.Recombinant antibodies expressed on the cell surface as Lpp-OmpA(46-159)fusions are functional and bind to antigens with high affinity. Suchfusions, manipulated as described below, will constitute preferred formsof the expression libraries of the present invention.

Mutagenesis

Where employed (i.e., directed evolution), mutagenesis will beaccomplished by a variety of standard, random mutagenic procedures.Mutation is the process whereby changes occur in the quantity orstructure of an organism. Mutation can involve modification of thenucleotide sequence of a single gene, blocks of genes or wholechromosome. Changes in single genes may be the consequence of pointmutations which involve the removal addition or substitution of a singlenucleotide base within a DNA sequence, or they may be the consequence ofchanges involving the insertion or deletion of large numbers ofnucleotides.

Mutations can arise spontaneously as a result of events such as errorsin the fidelity of DNA replication or the movement of transposablegenetic elements (transposons) within the genome. They are also inducedfollowing exposure to chemical or physical mutagens. Suchmutation-inducing agents include ionizing radiations, ultraviolet lightand a diverse array of chemical such as alkylating agents and polycyclicaromatic hydrocarbons all of which are capable of interacting eitherdirectly or indirectly (generally following some metabolicbiotransformations) with nucleic acids. The DNA lesions induced by suchenvironmental agents may lead to modifications of bas e sequence whenthe affected DNA is replicated or repaired and thus to a mutation.

Insertional Mutagenesis

Insertional mutagenesis is based on the inactivation of a gene viainsertion of a known DNA fragment. Because it involves the insertion ofsome type of DNA fragment, the mutations generated are generallyloss-of-function rather than gain-of-function mutations. However, thereare several examples of insertions generating gain-of-function mutations(Moncz et al. 1990; Oppenheimer et al. 1991). Insertion mutagenesis hasbeen very successful in bacteria and Drosophila (Cooley et al. 1988) andrecently has become a powerful tool in several plant species (corn;e.g., Schmidt et al. 1987); Arabidopsis; e.g., Herman and Marks 1989;Koncz et al. 1990; Antirrhinum: e.g., Sommer et al. 1990).

Transposable genetic elements are DNA sequences that can move(transpose) from one place to another in the genome of a cell. The firsttransposable elements to be recognized were the Activator/Dissociationelements of Zea mays (McClintock, 1957). Since then they have beenidentified in a wide range of organisms, both prokaryotic and eukaryotic(Berg and Howe 1989).

Transposable elements in the genome are characterized by being flankedby direct repeats of a short sequence of DNA that has been duplicatedduring transposition and is called a target site duplication. Virtuallyall transposable elements whatever their type, and mechanism oftransposition, make such duplications at the site of their insertion. Insome cases the number of bases duplicated is constant, in other cases itmay vary with each transposition event. Most transposable elements haveinverted repeat sequences at their termini these terminal invertedrepeats may be anything from a few bases to a few hundred bases long andin many cases they are known to be necessary for transposition.

Prokaryotic transposable elements have been most studied in E. coli andGram negative bacteria but are also present in Gram positive bacteria.They are generally termed insertion sequences if they are less thanabout 2 kilobases long or transposons if they are longer. Bacteriophagessuch as mu and D108 which replicate by transposition make up a thirdtype of transposable element elements of each type encode at least onepolypeptide a transposase, required for their own transposition.Transposons further often include genes coding for function unrelated totransposition and often carry antibiotic resistance genes.

Transposons can be divided into two classes according to theirstructure. Firstly, compound or composite transposons have copies of aninsertion sequence element at each end usually in an invertedorientation. These transposons require transposases to be encoded by oneof their terminal IS elements. The second class of transposon haveterminal repeats of about 30 base pairs and do not contain sequencesfrom IS elements.

Transposition is usually either conservative or replicative although insome cases it can be both. In replicative transposition one copy of thetransposing element remains at the donor site and another is inserted atthe target site. Conservative transposition the transposing element isexcised from one site and inserted at another.

Eukaryotic elements can also be classified according to their structureand mechanism of transportation. The primary distinction is betweenelements that transpose via an RNA intermediate and elements thattranspose directly from DNA to DNA.

Elements that transpose via an RNA intermediate are often referred to asretrotransposons and their most characteristic feature is that theyencode polypeptides that are believed to have reverse transcriptionaseactivity. There are two types of retrotransposon: some resemble theintegrated proviral DNA of a retrovirus in that they have long directrepeat sequences, long terminal repeats (LTRs), at each end. Thesimilarity between these retrotransposons and proviruses extends totheir coding capacity. They contain sequences related to the gag and polgenes of a retrovirus, suggesting that they transpose by a mechanismrelated to a retroviral life cycle. Retrotransposons of the second typehave no terminal repeats. They also code for gag- and pol-likepolypeptides and transpose by reverse transcription of RNA intermediatesbut do so by a mechanism that differs from that or retrovirus-likeelements. Transposition by reverse transcription is a replicativeprocess and does not require excision of an element from a donor site.

Transposable elements are an important source of spontaneous mutationsand must have influenced the ways in which genes and genomes haveevolved. They can inactivate genes by inserting within them and cancause gross chromosomal rearrangements either directly through theactivity of their transposases or indirectly as a result ofrecombination between copies of an element scattered around the genome.Transposable elements that excise often do so imprecisely and mayproduce alleles coding for altered gene products if the number of basesadded or deleted is a multiple of three.

Transposable elements themselves may evolve in unusual ways. If theywere inherited like other DNA sequences then copies of an element in onespecies would be more like copies in closely related species than copiesin more distant species. This is not always the case, suggesting thattransposable elements are occasionally transmitted horizontally from onespecies to another.

Chemical Mutagenesis

Chemical mutagenesis offers certain advantages, the ability to find afull range of mutant alleles with degrees of phenotypic severity, and isfacile and inexpensive to perform. The majority of chemical carcinogensproduce mutations in DNA. Benzo[a]pyrene, N-acetoxy-2-acetylaminofluorene and aflotoxin B1 cause GC to TA transversions in bacteriaand mammalian cells . Benzo[a]pyrene also can produce base substitutionssuch as AT to TA. N-nitroso compounds produce GC to AT transitions.Alkylation of the O4 position of thymine induced by exposure ton-nitrosoureas results in TA to CG transitions.

A high correlation between mutagenicity and carcinogenity is theunderlying assumption behind the Ames test (McCann et al., 1975incorporated herein by reference) which speedily assays for mutants in abacterial system, together with an added rat liver homogenate, whichcontains the microsomal cytochrome P450, to provide the metabolicactivation of the mutagens where needed.

In vertebrates several carcinogens have been found to produce mutationin the ras proto-oncogene. N-nitroso-N-methyl urea induces mammary,prostate and other carcinomas in rats with the majority of the tumorsshowing a G to A transition at the second position in codon 12 of theHa-ras oncogene. Benzo[a]pyrene-induced skin tumors contain A to Ttransformation in the second codon of the Ha-ras gene.

Radiation Mutagenesis

The integrity of biological molecules is degraded by the ionizingradiation. Adsorption of the incident energy leads to the formation ofions and free radicals, and breakage of some covalent bonds.Susceptibility to radiation damage appears quite variable betweenmolecules, and between different crystalline forms of the same molecule.It depends on the total accumulated dose, and also the dose rate (asonce free radicals are present, the molecular damage they cause dependson their natural diffusion rate and thus upon real time). Damage isreduced and controlled by making the sample as cold as possible.

Ionizing radiation causes DNA damage and cell killing, generallyproportional to the dose rate. Ionizing radiation has been postulated toinduce multiple biological effects by direct interaction with DNA orthrough the formation of free radical species leading to DNA damage(Hall, 1988). These effects include gene mutations, malignanttransformation, and cell killing. Although ionizing radiation has beendemonstrated to induce expression of certain DNA repair genes in someprokaryotic and lower eukaryotic cells, little is known about theeffects of ionizing radiation on the regulation of mammalian geneexpression (Borek, 1985). Several studies have described changes in thepattern of protein synthesis observed after irradiation of mammaliancells. For example, ionizing radiation treatment of human malignantmelanoma cells is associated with induction of several unidentifiedproteins (Boothman, et al., 1989). Synthesis of cyclin and co-regulatedpolypeptides is suppressed by ionizing radiation in rat REF52 cells butnot in oncogene-transformed REF52 cell lines (Lambert and Borek, 1988).Other studies have demonstrated that certain growth factors or cytokinesmay be involved in x-ray-induced DNA damage. In this regard,platelet-derived growth factor is released from endothelial cells afterirradiation (Witte, et al., 1989).

In the present invention, the term “ionizing radiation” means radiationcomprising particles or photons that have sufficient energy or canproduce sufficient energy via nuclear interactions to produce ionization(gain or loss of electrons). An exemplary and preferred ionizingradiation is an x-radiation. The amount of ionizing radiation needed ina given cell generally depends upon the nature of that cell. Typically,an effective expression-inducing dose is less than a dose of ionizingradiation that causes cell damage or death directly. Means fordetermining an effective amount of radiation are well known in the art.

In a certain embodiments, an effective expression inducing amount isfrom about 2 to about 30 Gray (Gy) administered at a rate of from about0.5 to about 2 Gy/minute. Even more preferably, an effective expressioninducing amount of ionizing radiation is from about 5 to about 15 Gy. Inother embodiments, doses of 2-9 Gy are used in single doses. Aneffective dose of ionizing radiation may be from 10 to 100 Gy, with 15to 75 Gy being preferred, and 20 to 50 Gy being more preferred.

Any suitable means for delivering radiation to a tissue may be employedin the present invention in addition to external means. For example,radiation may be delivered by first providing a radiolabeled antibodythat immunoreacts with an antigen of the tumor, followed by deliveringan effective amount of the radiolabeled antibody to the tumor. Inaddition, radioisotopes may be used to deliver ionizing radiation to atissue or cell.

Random and In Vitro Scanning Mutagenesis

Random mutagenesis may also be introduced using error prone PCR (Cadwelland Joyce, 1992). The rate of mutagenesis may be increased by performingPCR in multiple tubes with dilutions of templates.

Structure-guided site-specific mutagenesis represents a powerful toolfor the dissection and engineering of protein-ligand interactions(Wells, 1996, Braisted and Wells, 1996). One particularly usefulmutagenesis technique is alanine scanning mutagenesis in which a numberof residues are substituted individually with the amino acid alanine sothat the effects of losing side-chain interactions can be determined,while minimizing the risk of large-scale perturbations in proteinconformation (Cunningham et al., 1989).

Comprehensive information on the functional significance and informationcontent of a given residue of an antibody can best be obtained bysaturation mutagenesis in which all 19 amino acid substitutions areexamined. The shortcoming of this approach is that the logistics ofmultiresidue saturation mutagenesis are daunting (Warren et al., 1996,Brown et al., 1996; Harrison et al., 1996; Burton and Barbas, 1994;Yelton et al., 1995; Jackson et al., 1995; Short et al., 1995; Wong etal., 1996; Hilton et al., 1996). Hundreds, and possibly even thousands,of site specific mutants must be studies. For each mutant protein, theappropriate gene construct must be made, the DNA must be transformedinto a host organism, transformants need to be selected and screened forexpression of the protein, the cells have to be grown to produce theprotein, and finally the recombinant mutant protein must be isolated.There have been only a handful of studies where one, or at most a few,residues in an antibody have been subjected to saturation mutagenesis.Even in those studies, only some of the mutants were examined in detail(Ito et al., 1996, Chen et al., 1995, Brumell et al., 1993).

In recent years, techniques for estimating the equilibrium constant forligand binding using minuscule amounts of protein have been developed(Parker, 1978; Blackburn et al., 1991; U.S. Pat. Nos. 5,221,605 and5,238,808.). The inventors own work however, has shown that the abilityto perform functional assays with small amounts of material can beexploited to develop highly efficient, in vitro methodologies for thesaturation mutagenesis of antibodies. The inventors bypassed cloningsteps by combining PCR mutagenesis with coupled in vitrotranscription/translation for the high throughput generation of proteinmutants. Here, the PCR products are used directly as the template forthe in vitro transcription/translation of the mutant single chainantibodies. Because of the high efficiency with which all 19 amino acidsubstitutions can be generated and analyzed in this way, it is nowpossible to perform saturation mutagenesis on numerous residues ofinterest, a process that can be described as in vitro scanningsaturation mutagenesis (Burks et al., 1997).

In vitro scanning saturation mutagenesis provides a rapid method forobtaining a large amount of structure-function information including:(i) identification of residues that modulate ligand binding specificity,(ii) a better understanding of ligand binding based on theidentification of those amino acids that retain activity and those thatabolish activity at a given location, (iii) an evaluation of the overallplasticity of an active site or protein subdomain, (iv) identificationof amino acid substitutions that result in increased binding.

The surface-displayed antibodies molecules may further be presented asfusion proteins that include reporter molecules, e.g. alkalinephosphatase, luciferase, β-lactamase, green fluorescent protein andothers.

Enzyme Constructs

In addition to the fusion constructions described above, vectors forenzyme expression require that appropriate signals be provided for thesynthesis of mRNA and polypeptides, and include various regulatoryelements such as enhancers/promoters from both bacterial and eukaryoticsystems that drive expression of the enzymes of interest in theappropriate host cells. Elements designed to optimize messenger RNAstability and translatability in host cells also are defined. Theconditions for the use of a number of dominant drug selection markersfor establishing permanent, stable cell clones expressing the productsare also provided, as is an element that links expression of the drugselection markers to expression of the polypeptide.

(i) Regulatory Elements

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. In preferred embodiments, thenucleic acid encoding a gene product is under transcriptional control ofa promoter. A “promoter” refers to a DNA sequence recognized by thesynthetic machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a gene. The phrase“under transcriptional control” means that the promoter is in thecorrect location and orientation in relation to the nucleic acid tocontrol RNA polymerase initiation and expression of the gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

In prokaryotic gene expression, unlike eukaryotic systems the DNA is notseparated from the cytoplasm by a nuclear membrane. There are also manyother differences in mRNA processing of prokaryotes and eukaryotes. Thefirst control point of prokaryotic gene expression is initiation oftranscription. Transcription is initiated at positions defined preciselyby promoters. Analysis of more than 100 promoters in E. coli hasidentified two consensus sequences positioned 350 and 10 base pairsupstream of the point in the DNA sequence where transcription begins.These sequences are involved in polymerase recognition and binding.

The topology of promoter DNA is also important, numerous bacterialpromoters are known to those of skill in the art (Makrides, 1996,incorporated herein by reference). The coiling of the double helixbrings the two sequence motifs into the correct position for efficientrecognition and binding of RNA polymerase. In some promoters, initiationof transcription is regulated by changes in the degree of supercoilingof the DNA (Lilley and Higgins, 1991). The RNA polymerase is amulti-subunit enzyme; the core enzyme is capable of transcriptionalelongation on its own but requires the addition of a further subunit (σ)in order to bind specifically to promoter sites and initiatetranscription. The synthesis of σ factors in prokaryotes allows thepolymerase to be directed to different sets of promoters (Helmann andChamberlain, 1988). In B. subtilis, for example, several different σfactors are produced at different stages so that different sets of genescan be turned on at each stage.

In the absence of ancillary proteins the rate of initiation varies by upto a factor of at least a 1000 over the whole range of promoters in E.coli. The efficiency of initiation is related to the sequence andtopology of the promoter region. Gene expression occurring in theabsence of regulatory factors is known as constitutive. At manypromoters, transcriptional initiation may be increase by binding tospecific regulatory proteins.

A feature of gene organization common to prokaryotes but rare ineukaryotes is the grouping of functionally related genes into operons.In an operon, genes encoding, for example the different enzymes of ametabolic pathway are clustered and are transcribed together into apolycistronic transcript, under the control of a single promoter. Thistranscript is then translated to give individual proteins. Operonsenable the rapid and efficient coordinate expression of a set of genesrequired to respond to a change in the external or internal environmentof the microorganism. Premature termination plays a part in theregulation of expression of operons.

The termination of transcription occurs at specific sites. There are twotypes of termination events. The first (factor independent termination)occurs at sites defined by a series of U residues preceded by aninverted repeat that forms a stem-loop structure at the 3′ end of theRNA transcript. This structure interferes with the polymerase action andleads to the release of the RNA. Factor dependent termination isdependent on the interaction of protein factor rho p with the RNApolymerase.

In eukaryotic systems, at least one module in each promoter functions toposition the start site for RNA synthesis. The best known example ofthis is the TATA box, but in some promoters lacking a TATA box, such asthe promoter for the mammalian terminal deoxynucleotidyl transferasegene and the promoter for the SV40 late genes, a discrete elementoverlying the start site itself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the thymidine kinase promoter, thespacing between promoter elements can be increased to 50 bp apart beforeactivity begins to decline. Depending on the promoter, it appears thatindividual elements can function either co-operatively or independentlyto activate transcription.

The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of direction the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent to and under thecontrol of a promoter that is capable of being expressed in a humancell. Generally speaking, such a promoter might include either a humanor viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, rat insulin promoter and glyceraldehyde-3-phosphatedehydrogenase can be used to obtain high-level expression of the codingsequence of interest. The use of other viral or mammalian cellularpromoters which are well-known in the art to achieve expression of acoding sequence of interest is contemplated as well, provided that thelevels of expression are sufficient for a given purpose.

By employing a promoter with well-known properties, the level andpattern of expression of the protein of interest following transfectionor transformation can be optimized. Further, selection of a promoterthat is regulated in response to specific physiologic signals can permitinducible expression of the gene product. Tables 2 and 3 list severalelements/promoters which may be employed, in the context of the presentinvention, to regulate the expression of the gene of interest. This listis not intended to be exhaustive of all the possible elements involvedin the promotion of gene expression but, merely, to be exemplarythereof.

One may include a polyadenylation signal to effect properpolyadenylation of the gene transcript in eukaryotic cells. The natureof the polyadenylation signal is not believed to be crucial to thesuccessful practice of the invention, and any such sequence may beemployed such as human growth hormone and SV40 polyadenylation signals.Also contemplated as an element of the expression cassette is aterminator. These elements can serve to enhance message levels and tominimize read through from the cassette into other sequences.

(ii) Selectable Markers

In certain embodiments of the invention, the cells contain nucleic acidconstructs of the present invention, a cell may be identified in vitroor in vivo by including a marker in the expression construct. Suchmarkers would confer an identifiable change to the cell permitting easyidentification of cells containing the expression construct. Usually theinclusion of a drug selection marker aids in cloning and in theselection of transformants, for example, genes that confer resistance toampicillin, tetracycline, neomycin, puromycin, hygromycin, DHFR, GPT,zeocin and histidinol are useful selectable markers. Alternatively,enzymes such as herpes simplex virus.

(iii) Multigene Constructs and IRES

In certain embodiments of the invention, the use of internal ribosomebinding sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picanovirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter to transcribe a single message.

Any heterologous open reading frame can be linked to IRES elements. Thisincludes genes for secreted proteins, multi-subunit proteins, encoded byindependent genes, intracellular or membrane-bound proteins andselectable markers. In this way, expression of several proteins can besimultaneously engineered into a cell with a single construct and asingle selectable marker.

Antibody and Antibody Fragment Constructs

Using the fusion technology described above, the present invention alsocontemplates the generation of host cells expressing, on their surface,antibodies or antibody fragments representing a library of antibodiesproduced in response to one or more immunogens. “Antibody” or “antibodyfragment” refers to any immunologic binding agent such as IgG, IgM, IgA,IgD and IgE or any antibody-like molecule that has an antigen bindingregion, and includes antibody fragments such as Fab′, Fab, F(ab′)₂,single domain antibodies (DABs), Fv, scFv (single chain Fv) and thelike. The techniques for preparing and using various antibody-basedconstructs and fragments are well known in the art.

The specificity of an antibody is determined by the complementaritydetermining regions (CDRs) within the light chain variable regions(V_(L)) and heavy chain variable regions (V_(H)). The F_(ab) fragment ofan antibody, which is about one-third the size of a complete antibodycontains the heavy and light chain variable regions, the complete lightchain constant region and a portion of the heavy chain constant region.F_(ab) molecules are stable and associate well due to the contributionof the constant region sequences. However, the yield of functionalF_(ab) expressed in bacterial systems is lower than that of the smallerF_(v) fragment which contains only the variable regions of the heavy andlight chains. The F_(v) fragment is the smallest portion of an antibodythat still retains a functional antigen binding site. The F_(v) fragmenthas the same binding properties as the F_(ab), however without thestability conferred by the constant regions, the two chains of the F_(v)can dissociate relatively easily in dilute conditions.

To overcome this problem, V_(H) and V_(L) regions may be fused via apolypeptide linker (Huston et al., 1991) to stabilize the antigenbinding site. This single polypeptide F_(v) fragment is known as asingle chain antibody (scF_(v)). The V_(H) and V_(L) can be arrangedwith either domain first. The linker joins the carboxy terminus of thefirst chain to the amino terminus of the second chain.

While the present invention has been illustrated with display of singlechain Fv molecules on the surface of the bacteria, one of skill in theart will recognize that heavy or light chain Fv or Fab fragments mayalso be used with this system. A heavy or light chain can be displayedon the surface followed by the addition of the complementary chain tothe solution. The two chains are then allowed to combine on the surfaceof the bacteria to form a functional antibody fragment. Addition ofrandom non-specific light or heavy chain sequences allows for theproduction of a combinatorial system to generate a library of diversemembers.

Antibody and Antibody Fragment Gene Isolation

To accomplish construction of antibodies and antibody fragments, theencoding genes are isolated and then modified to permit cloning into theexpression vector. Although methods can be used such as probing the DNAfor V_(H) and V_(L) from hybridoma cDNA (Maniatis et al., 1982) orconstructing a synthetic gene for V_(H) and V_(L) (Barbas et al., 1992),a convenient mode is to use template directed methods to amplify theantibody sequences. A diverse population of antibody genes can beamplified from a template sample by designing primers to the conservedsequences at the 3′ and 5′ ends of the variable region known as theframework or to the constant regions of the antibody (Iverson et al,1989). Within the primers, restriction sites can be placed to facilitatecloning into an expression vector. By directing the primers to theseconserved regions, the diversity of the antibody population ismaintained to allow for the construction of diverse libraries. Thespecific species and class of antibody can be defined by the selectionof the primer sequences as illustrated by the large number of sequencesfor all types of antibodies given in Kabat et al., 1987, herebyincorporated by reference.

Messenger RNA isolated from the spleen or peripheral blood of an animalcan be used as the template for the amplification of an antibodylibrary. In certain circumstances, where it is desirable to display ahomogeneous population of antibody fragments on the cell surface, mRNAmay be isolated from a population of monoclonal antibodies. MessengerRNA from either source can be prepared by standard methods and useddirectly or for the preparation of a cDNA template. Generation of mRNAfor cloning antibody purposes is readily accomplished by following thewell-known procedures for preparation and characterization of antibodies(see, e.g., Antibodies: A Laboratory Manual, 1988; incorporated hereinby reference).

Method of Producing Monoclonal Antibodies

Generation of monoclonal antibodies (MAbs) follows generally the sameprocedures as those for preparing polyclonal antibodies. Briefly, apolyclonal antibody is prepared by immunizing an animal with animmunogenic composition in accordance and collecting antisera from thatimmunized animal. A wide range of animal species can be used for theproduction of antisera. Typically the animal used for production ofanti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or agoat. Because of the relatively large blood volume of rabbits, rabbitsare usually preferred for production of polyclonal antibodies.

Immunogenic compositions often vary in immunogenicity. It is oftennecessary therefore to boost the host immune system, as may be achievedby coupling a peptide or polypeptide immunogen to a carrier. Exemplaryand preferred carriers are keyhole limpet hemocyanin (KLH) and bovineserum albumin (BSA). Other albumins such as ovalbumin, mouse serumalbumin or rabbit serum albumin can also be used as carriers. Recognizedmeans for conjugating a polypeptide to a carrier protein are well knownand include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimideester, carbodiimides and bis-diazotized benzidine.

The immunogenicity of a particular immunogen composition may be enhancedby the use of non-specific stimulators of the immune response, known asadjuvants. Exemplary and preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster injection, may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated, stored and thespleen harvested for the isolation of mRNA from the polyclonal responseor the animal can be used to generate MAbs for the isolation of mRNAfrom a homogeneous antibody population.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g. a small moleculehapten conjugated to a carrier, a purified or partially purifiedprotein, polypeptide or peptide. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. Rodents such as mice and rats are frequently used animals;however, the use of rabbit, sheep frog cells is also possible. The useof rats may provide certain advantages (Goding, pp. 60-61, 1986), butmice are preferred, particularly the BALB/c mouse as this is mostroutinely used and generally gives a higher percentage of stablefusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from blood samples. Spleencells and blood cells are preferable, the former because they are a richsource of antibody-producing cells that are in the dividing plasmablaststage, and the latter because blood is easily accessible. Often, a panelof animals will have been immunized and the spleen of animal with thehighest antibody titer will be removed and the spleen lymphocytesobtained by homogenizing the spleen with a syringe. Typically, a spleenfrom an immunized mouse contains approximately 5×10⁷ to 2×10⁸lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986; Campbell, 1984). Forexample, where the immunized animal is a mouse, one may use P3-X63/Ag8,X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3,IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 areall useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler & Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al., 1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp.71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. Simple and rapid assaysinclude radioimmunoassays, enzyme immunoassays, cytotoxicity assays,plaque assays, dot immunobinding assays, and the like.

The selected hybridomas are serially diluted and cloned into individualantibody-producing cell lines from which clones can then be propagatedindefinitely to provide MAbs. The cell lines may be exploited for MAbproduction in two basic ways. A sample of the hybridoma can be injected(often into the peritoneal cavity) into a histocompatible animal of thetype that was used to provide the somatic and myeloma cells for theoriginal fusion. The injected animal develops tumors secreting thespecific monoclonal antibody produced by the fused cell hybrid. The bodyfluids of the animal, such as serum or ascites fluid, can then be tappedto provide MAbs in high concentration. The individual cell lines couldalso be cultured in vitro, where the MAbs are naturally secreted intothe culture medium from which they can be readily obtained in highconcentrations. MAbs produced by either means may be further purified,if desired, using filtration, centrifugation and various chromatographicmethods such as HPLC or affinity chromatography.

Following the isolation and characterization of the desired monoclonalantibody, the mRNA can be isolated using techniques well known in theart and used as a template for amplification of the target sequence.

Amplification of Gene Fragments

A number of template dependent processes are available to amplify thetarget sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (PCR™),which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, and in Innis et al. (1990), each of which is incorporatedherein by reference in its entirety. Briefly, in PCR™, two primersequences are prepared which are complementary to regions on oppositecomplementary strands of the target sequence. An excess ofdeoxynucleoside triphosphates are added to a reaction mixture along witha DNA polymerase, e.g., Taq polymerase. If the target sequence ispresent in a sample, the primers will bind to the target and thepolymerase will cause the primers to be extended along the targetsequence by adding on nucleotides. By raising and lowering thetemperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction products and the process isrepeated. Preferably a reverse transcriptase PCR™ amplificationprocedure may be performed in order to quantify the amount of targetamplified. Polymerase chain reaction methodologies are well known in theart.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPA No. 320 308, incorporated herein by reference in itsentirety. In LCR, two complementary probe pairs are prepared, and in thepresence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR™, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as an amplification method. In this method, a replicativesequence of RNA which has a region complementary to that of a target isadded to a sample in the presence of an RNA polymerase. The polymerasewill copy the replicative sequence which can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids (Walker et al., 1992).

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR) involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases can be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencescan also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having a 3′ and 5′ sequences of non-specific DNA and middlesequence of specific RNA is hybridized to DNA which is present in asample. Upon hybridization, the reaction is treated with RNaseH, and theproducts of the probe identified as distinctive products which arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Other amplification methods are described in GB Application No. 2 202328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR™ like, template and enzymedependent synthesis. The primers may be modified by labeling with acapture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes is added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989). In NASBA, the nucleicacids can be prepared for amplification by standard phenol/chloroformextraction, heat denaturation of a clinical sample, treatment with lysisbuffer and minispin columns for isolation of DNA and RNA or guanidiniumchloride extraction of RNA. These amplification techniques involveannealing a primer which has target specific sequences. Followingpolymerization, DNA/RNA hybrids are digested with RNase H while doublestranded DNA molecules are heat denatured again. In either case thesingle stranded DNA is made fully double stranded by addition of secondtarget specific primer, followed by polymerization. The double strandedDNA molecules are then multiply transcribed by a polymerase such as T7or SP6. In an isothermal cyclic reaction, the RNAs are reversetranscribed into double stranded DNA, and transcribed once against witha polymerase such as T7 or SP6. The resulting products, whethertruncated or complete, indicate target specific sequences.

Davey et al., EPA No. 329 822 (incorporated herein by reference in itsentirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting as a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “race” and “one-sidedPCR” (Frohman, 1990; O'Hara et al., 1989).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step (Wu et al., 1989).

Amplification products may be analyzed by agarose, agarose-acrylamide orpolyacrylamide gel electrophoresis using standard methods (see, e.g.,Maniatis et al. 1982). For example, one may use a 1% agarose gel stainedwith ethidium bromide and visualized under UV light. Alternatively, theamplification products may be integrally labeled with radio- orfluorometrically-labeled nucleotides. Gels can then be exposed to x-rayfilm or visualized under the appropriate stimulating spectra,respectively.

Semisynthetic Antibody Gene Fragments and Preparation of Mutants

Genes for antibody fragments also may be generated by semisyntheticmethods known in the art (Barbas et al., 1992). Using the conservedregions of an antibody fragment as a framework, variable regions can beinserted in random combinations one or more at a time to alter thespecificity of the antibody fragment and generate novel binding sites,especially in the generation of antibodies to antigens not conducive toimmunization such as toxic or labile compounds. Along the same lines aknown antibody sequence may be varied by introducing mutations randomlyor site specifically. This may be accomplished by methods well known inthe art such as mutagenesis with mismatched primers or error-prone PCR™(Innir, 1990).

D. Host Cells, Methods of Producing Host Cells and “Dead Man” Selection

Virtually any cell may be used as a host cell, but the ease with whichbacterial cell are handled makes these a preferred embodiment, at leastwhere eukaryotic modification events (glycosylation, etc.) are notnecessary. For example, mammalian cells, plant cells and yeast cells allmay be employed. Preferred bacterial hosts include Gram negativebacterial cells, particularly E. coli, although Salmonella, Klebsiella,Erwinia, Pseudomonas aeruginosa, Haemophilus influenza, Rickettsiarickettsii, Neisseria gonorrhea, etc. also are suitable.

Bacterial cells in which polypeptides are expressed may be readilyimmobilized, thus allowing rapid recovery and efficient removal from thesystem. One may use membranes, dipsticks or beads through a chemicallypromoted coupling reaction in addition to other well-knownimmobilization matrices. In this manner, the cells can be separated fromthe solution without the need for a centrifugation step.

Bacterial cultures may be supplied in forms that have an indefiniteshelf life and yet can be readily prepared for use; for example as “stabcultures” or lyophilized preparations; the user may prepare largeamounts in liquid culture as needed. The reagent is thus renewable ascompared with other polypeptide reagents that are “used up” and must bereplaced continually. Bacterial cultures may be prepared fresh withoutconcern about shelf-life of reagents that must be stored until use.

Selection

Growth studies of bacteria have demonstrated that the viability ofpositive bacteria cells (with fusion proteins) and negative cells(without fusion proteins or with truncated fusion proteins) varysignificantly. The quantity of negative cells overwhelm the positivecells after the required 12 to 24 hours growth. For example, thepercentage of positive cells in a library may decrease 10 to 100 foldduring the growth period. Since most FACS are optimally designed forsorting mammalian cells which are 10 to 100 times larger than bacterialcell, even using the most stringent selection mode, the negative cellsmay be selected along with the positive cells.

Elimination of these negative cells in a presort library is a veryimportant step for a successful sorting experiment. A number ofapproaches may be used to eliminate negative cells.

Negative Selection Systems

The blue-white screening system has been widely used for libraryscreening in molecular biology. In this system, a β-galactosidase enzymeis used as a reporter protein (Maniatis, 1989), and a multiple cloningsite (MCS) is located in the middle of the β-galactosidase gene. Aninsertion of DNA into the MCS may result in the interruption of theβ-galactosidase gene. Thus, these cells with the DNA insert will not beable to produce an active β-galactosidase, and consequently cannotconvert the substrate (X-gal) into a colored product, while the cellswithout an insert can produce β-galactosidase and hydrolyze thesubstrate into the indigo colored product. This allows for selection ofcolorless colonies which usually have an insert are picked and streakedonto a new plate. The process is repeated until a plasmid carrying a DNAinsert is confirmed. Finding a positive colony with the right insert mayrequire several rounds of selections. Since the selection is performedon an agar plate, the speed of screening is not suitable for highthroughput screening (for example, millions of colonies per day).

Positive Selection Systems

The direct selection of an antibody with enhanced catalytic activity oraltered specificity is possible when the reaction of interest can becoupled to the growth or survival of a cell, such as the release of anessential nutrient or cofactor. However, many reactions of interest donot easily lend themselves to such a selection scheme. In addition,prokaryote metabolism is very complex, and bacteria are usually capableof adapting to new metabolic pathways, resulting in a large number offalse positives (Lesley, 1993).

A variety of positive selection vectors have been developed to preventthe existence of the cells carrying non-insert plasmids. All thestrategies rely on the inactivation of either a lethal gene (O'Connor1982, Henrich 1986, Kuhn 1986, Arakawa 1991, Bernard 1994), a lethalsite (Hagan 1982), a dominant function conferring cell sensitivity tometabolites (Dean 1981, Burns 1984, Pierce 1992, Gossen 1992) or arepressor of an antibiotic-resistance function (Robert 1980, Nikolnikov1984). Most of these strategies are not well adapted for general use dueto their large size, the limited number of cloning sites, or the need ofspecial host strain and special culture medium. Another problem of thesesystems is that they are not an expression system, and the inserted genecannot be expressed unless it carries a complete promoter and operatorsequence.

Seehaus has fused scFv DNA into the gene encoding β-lactamase (Bla) atthe 3′-terminus of the signal sequence. Only those clones carryinginserts that are in frame with Bla gene can survive ampicillinselection, while others that carry out-of-frame deletions or internalstop codons are eliminated. This strategy can be applied to theGeorgiou/Iverson system, however, the size of the fusion protein Bla (58to 60 Kda) creates another problem when the strategy is implemented.Only small proteins (<53 Kda) can be displayed on the surface of E. coliand still retain significant activity (Stathopulos 1996). A1pp-OmpA-scFv-Bla Fusion protein is too big to be displayed on thesurface of bacteria. Nevertheless, this general resistance fusionstrategy can be modified for our purposes.

The inventors have employed new positive (“dead man”) selection systemsin the present invention using two approaches to eliminate negativecells. In the first approach, the target gene is fused with part of anantibiotic gene and used as an insert. Without the insert, the cloningvector does not process the specific antibiotic resistance, and thecells having the self-ligated vectors are eliminated by antibioticselection. Two different plasmids were constructed with two antibioticmarkers, penicillin resistance and chloramphenicol resistance, as wellas the surface expression 1pp-OmpA machinery (same as pTX152 which canexpress the scFv on the surface of E. coli).

In one embodiment a plasmid was constructed to eliminate cells withoutsurface-expressed scFv. A restriction site was introduced in the middleof the chloramphenicol acetyl transferase (CAT) gene and in the middleof scFv gene. The N-terminal part of CAT (200bps) was fused to the endof scFv gene by overlapping PCR, while the cloning vector carried theC-terminal part of antibiotic gene. The cells that contain a plasmidwithout the appropriate insert (for example, antibody gene andN-terminal part of CAT) cannot express functional CAT protein, so theyare eliminated in a chloramphenicol medium.

In another embodiment, a plasmid is constructed to eliminate cells thatcarry a advertition stop codon in the middle of the scFv gene. Thepromoter and ribosome binding site (rbs) for CAT are eliminated, andboth surface expression 1pp-OmpA-scFv gene and CAT gene are undercontrol of a single promoter and operator. Thus, the CAT gene can betranscribed but cannot be translated due to the lack of a rbs. The scFvgene and the CAT gene are fused in such a way that the stop codon of thescFv and the start codon of the CAT are arranged in the order of TAATG.When the ribosome stop at the TAA of scFV gene, it can frameshift acertain fraction of the time to the adjacent ATG codon thus restart thetranslation of CAT gene (Benhar). The stop codons caused by anunexpected mutation in the middle of the scFv gene will force theribosome to fall off the mRNA early, so it will not be able to translatethe CAT gene. As a result, the cells that carry the stop codon(s) in themiddle of scFv gene do not have chloramphenicol resistance and are notable to survive in chloramphenicol medium.

It is envisioned that analogous cloning vectors also might improve theconstruction and screening of phage display libraries by reducing thenumber of non-insert plasmids in the presort antibody libraries, andthereby reduce the number of selection rounds required.

Of course, it is understood that other antibiotic resistance genes suchas the ampicillin resistance gene and kanamycin resistance gene can befused to the antibody gene in this system. Furthermore, any gene productthat is essential for bacterial growth may be used.

Gene Transfer

Technology for introduction of DNA into cells is well-known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (i) chemical methods such as calcium phosphateprecipitation (Graham et al., 1973; Zatloukal et al., 1992); (ii)physical methods such as protoplast fusion, microinjection (Capecchi,1980), electroporation (Wong et al., 1982; Fromm et al., 1985; U. S.Patent No. 5,384,253) and the gene gun (Johnston et al., 1994; Fynan etal., 1993); (iii) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitiset al., 1988a; 1988b); and (iv) receptor-mediated mechanisms (Curiel etal., 1991; 1992; Wagner et al., 1992).

E. Methods For Detecting Analytes Using Cells Displaying Scfv Antibodies

For the first time, a competitive immunoassay has been developed thattakes advantage of anti-analyte binding antibodies immobilized on abacterial cell surface. Several advantages of the disclosed immunoassayinclude generally the convenience, wide applicability and thesimplicity, rapidity and sensitivity of the assay. In addition, selectedhost cells displaying an polypeptide of interest may be used tostimulate an immune response.

The first step in the development of whole cells suitable for thedetection of analytes is the screening of antibody libraries displayedon the cell surface. Cells displaying antibodies having affinity for adesired analyte are isolated. First, a library of cell surface displayedproteins is prepared as described elsewhere in the specification. Forexample a library of surface displayed scFv antibodies can be preparedas described in Example 7. Once an expression library has been prepared,the selected antigen for which one desires to identify and isolatespecific antibody or antibodies is labeled with a detectable label.There are many types of detectable labels, including fluorescent labels,the latter being preferred in that they are easily handled, inexpensiveand non-toxic. The labeled antigen is contacted with the cellsdisplaying the antibody expression library under conditions that allowspecific antigen-antibody binding. Conditions can be varied so that onlyvery tightly binding interactions occur; for example, by using very lowconcentrations of labeled antigen.

Identifying the antibody or antibody fragment expressing cells may beaccomplished by methods that depend on detecting the presence of thebound detectable label. A particularly preferred method foridentification and isolation is cell sorting or flow cytometry. Oneaspect of this method is fluorescence activated cell sorting (FACS).

Following selection of high affinity clones, the production of solubleantibodies can be achieved easily without the need for furthersubcloning steps. Thus, the clones may be maintained under standardculture conditions and employed to produce the selected antibody.Production of antibody is limited only to the scaleup of the cultures.

The invention further includes competitive binding assays using cellswith antibodies or analyte-combining antibody fragments expressed on theouter cell surface. As used in the context of the present invention,analyte is defined as a species that interacts with a non-identicalmolecule to form a tightly bound, stable complex. For practicalpurposes, the binding affinity is usually greater than about 10⁶ M⁻¹ andis preferably in the range of 10⁹-10¹⁵ M⁻¹. The analyte may be any ofseveral types of organic molecules, including alicyclic hydrocarbons,polynuclear aromatics, halogenated compounds, benzenoids, polynuclearhydrocarbons, nitrogen heterocyclics, sulfur heterocyclics, oxygenheterocyclics, and alkane, alkene alkyne hydrocarbons, etc. Biologicalmolecules are of particular interest, including amino acids, peptides,proteins, lipids, saccharides, nucleic acids and combinations thereof.Of course it will be understood that these are by way of example onlyand that the disclosed immunoassay methods are applicable to detectingan extraordinarily wide range of compounds, so long as one can obtain anantibody that binds with the analyte of interest.

The disclosed whole cell immunoassay methods allow rapid detection of awide range of analytes and are particularly useful for determination ofpolypeptides. The methods have been developed to take advantage of thebinding characteristics of bacterial cell surface exposed anti-analyteantibodies. Such surface displayed antibodies are stable and bindreadily with specific analytes. This unique form of protein expressionand immobilization thus has provided the basis of an extremely rapidcompetitive assay that may be performed in a single reaction vessel inan “add and measure” format. Such assays can be described as “one-pot”reactions that make possible in situ detection of an analyte.

A particular advantage of cell surface expressed antigen-bindingantibodies is that the antibody is attached to the outer membrane of thecell. The cells therefore act as a solid support during the assay,thereby eliminating many of the manipulations typically required inpreparing reagents required for existing immunoassay techniques.Optionally, cells with the antibody displayed on the surface maythemselves be attached to a solid support such as a membrane, dipstickor beads to further facilitate removal of the cells following the assay.

The immunoassays of the present invention may be used to quantitate awide range of analytes. Generally, one first obtains the appropriatehost cell culture where the anti-analyte antibody is displayed on thehost cell surface, calibrates with standard samples of analyte, thenruns the assay with a measured volume of unknown concentration ofanalyte.

In conducting a competitive immunoassay in accordance with the disclosedmethods, one first obtains a host cell that expresses an analyte bindingantibody. The host cell is then contacted with a standard analyte samplethat contains a known amount of an analyte linked to a detectable labelemploying conditions effective for forming an immune complex. Oncecalibration is completed, the same procedure is used with a second hostcell that has the same antibody or analyte-combining fragment expressedon its surface, except that in addition to the standard labeled analytesample, a test sample in which an unknown amount of analyte is to bedetermined is added. One is not limited to using the same host cell inthis procedure.

In the actual assay, a known amount of the antibody-covered cells areplaced in a solution of a known concentration of the analyte-conjugatealong with an unknown concentration of the analyte (the test solution).The analyte conjugate competes with free analyte in solution for bindingto the antibody molecules on the cell surface. The higher theconcentration of analyte conjugate in the solution, the fewer moleculesof fluorescein analyte conjugate bind on the surface of the cells, andvice versa.

The mixture is centrifuged to pellet the cells, and the fluorescence ofthe supernatant is measured. The assay is quantitative because theamount of observed fluorescence is proportional to the concentration ofanalyte in the test sample, i.e., if there is a very low concentrationof analyte to compete with the fluorescein conjugate, then most of theconjugate will bind to the cells and will be removed from solution. Themore molecules of analyte in solution, the more molecules of analytebind to the antibodies thereby preventing the conjugate from binding. Inthis case, more fluorescein conjugate remains in the supernatant to givea stronger fluorescence signal. The assay can be calibrated to generatea quantitative measurement of the unknown concentration of analyte. Theentire assay requires less than one hour. Fluorescence determinationsmay be made with a basic fluorimeter.

The present invention involves a novel method of carrying outcompetitive immunoassays using antibodies attached to the surface ofcells. The disclosed immunoassays are useful for binding, purifying,removing, quantifying or otherwise generally detecting analytes.Antibodies expressed on bacterial cell surfaces have been shownsurprisingly adaptable for use in competitive immunoassay procedures. Ina particular example, the analyte digoxin, a cardiac glycoside, wasdetermined using fluorescein-digoxin conjugate. The assay wasquantitative with a sensitivity in the nanomolar range.

Cells expressing an antibody fragment on their surface may also belinked to a solid support, such as in the form of beads, membrane or acolumn matrix, and the sample suspected of containing the unwantedantigenic component applied to the immobilized antibody. A purged orpurified sample is then obtained free from the unwanted antigen simplyby collecting the sample from the column and leaving the antigenimmunocomplexed to the immobilized antibody.

Detection of immunocomplex formation is well known in the art and may beachieved through the application of numerous approaches. Theseapproaches are typically based upon the detection of a label or marker,such as any of the radioactive, fluorescent, chemiluminescent,electrochemiluminescent, biological or enzymatic tags or labels known inthe art. U.S. patents concerning the use of such labels include U.S.Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149 and 4,366,241, each incorporated herein by reference. Ofcourse, one may find additional advantages through the use of asecondary binding ligand such as a second antibody or a biotin/avidinligand binding arrangement, as is known in the art.

The first added component that becomes bound within the primary immunecomplexes may be detected by means of a second binding ligand that hasbinding affinity for the surface expressed antibody. In these cases, thesecond binding ligand may be linked to a detectable label. The secondbinding ligand is itself often an antibody, which may thus be termed a“secondary” antibody. The primary immune complexes are contacted withthe labeled, secondary binding ligand, or antibody, under conditionseffective and for a period of time sufficient to allow the formation ofsecondary immune complexes. The secondary immune complexes are thengenerally washed to remove any non-specifically bound labeled secondaryantibodies or ligands, and the remaining label in the secondary immunecomplexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the surface expressed antibody is used to formsecondary immune complexes, as described above. After washing, thesecondary immune complexes are contacted with a third binding ligand orantibody that has binding affinity for the second antibody, again underconditions effective and for a period of time sufficient to allow theformation of immune complexes (tertiary immune complexes). The thirdligand or antibody is linked to a detectable label, allowing detectionof the tertiary immune complexes thus formed. This system may providefor signal amplification if this is desired.

The competitive immunoassay discussed above requires one to set up acontrol sample. This sample comprises a second host cell expressing ananalyte binding antibody. This is contacted with a known amount ofanalyte linked to a detectable label and a known amount of unlabeledanalyte, i.e. the analyte to be detected or determined in the assay.After formation of the immunocomplexes, one measures the free label insolution after the cells have been separated. This measures the amountof residual detectable label as the decrease in, e.g., fluorescenceemission, from which the amount of unknown analyte may be determined. Apreferred fluorescent label is fluorescein. The inventors have foundthat measurement of the amount of residual label that is not bound toantibodies is proportional to the amount of analyte label in solution.This provides the basis for quantitative measure in that the increase inthe amount of label is directly proportional to the analyte.Alternatively, the label may be measured in the complexes. In this typeof measurement, the analyte is inversely proportional to the amount oflabel.

Of course fluorescence labeling, while preferred, does not preclude theuse of other detectable agents such as chemiluminescent agents,electrochemiluminescent agents, radioactive labels, enzymatic labelsthat form a colored product with a chromogenic substrate as well asother fluorescent compounds. A preferred fluorescent label isfluorescein while Ru(bpy)₃ ²⁺ is preferred for use as anelectrochemiluminescent agent.

The invention is readily adaptable to the determination of multipleanalytes. This is achieved using two or more different analyte-bindingantibodies expressed in separate host cells. It is also possible tosurface express more than one antibody on the surface of a particularhost cell; however, this may cause interference in binding. One will, inthese situations, use different detecting agents; for example, twodifferent fluorescent labels, each with distinct emissions, such asfluorescein which emits at 520 nm and Texas Red which emits at 620 nm.

In certain uses of the assay, cells containing antibodies on the surfaceare produced as described previously. Antibodies known to bind tightlyand specifically to a molecule of interest are employed. The moleculecould be a medically relevant molecule, a marker molecule used in ascientific study, a pesticide of environmental concern in groundwater,etc. A covalent conjugate of the molecule with a fluorescent moiety suchas fluorescein is synthesized for use as a probe in binding. Otherdetection agents include radioactive compounds, enzyme conjugates,chemiluminescent reagents such as luciferase and electrochemiluminescentreagents such as Ru(bpy)₃ ²⁺. (Yang et al., 1994; Blackburn et al,1991). The assay may also be carried out using tritium as the labelingagent for the antigen and performing a radioimmunoassay. Theradioactivity may be detected using a scintillation counter to measurebinding constants up to 10⁻⁸ or 10⁻¹.

To perform the preferred assay, a known amount of the antibody-coveredcells are placed in a solution of a known concentration of themolecule-fluorescein conjugate along with an unknown concentration ofthe molecule. The molecule-fluorescein conjugate competes with freemolecules in solution for binding to the antibody on the cell surface.The higher the concentration of the molecule in the solution, the fewermolecules of fluorescein-molecule conjugate will bind to the surface ofthe cells, while lower concentrations of the molecule will result inmore of the conjugate bound to the cell surface.

Before measuring residual fluorescence, the cells are removed from thesolution, most conveniently by pelleting, and the fluorescence of thesupernatant measured. The assay is quantitative because the amount ofobserved fluorescence is proportional to the concentration of themolecule in the unknown sample. If there is a very low concentration ofthe molecule to compete with the fluorescein conjugate, most of theconjugate will bind to the cells and will be removed from the solution.The higher the concentration of the molecule in solution, the moremolecules bind to the antibodies thereby preventing the conjugate frombinding. In this case, more fluorescein conjugate remains in thesupernatant to give a stronger fluorescent signal. The assay can becalibrated to generate a quantitative measurement of the unknownconcentration of the molecule. The entire assay takes less than an hourand requires only a basic fluorimeter.

As part of the invention, immunoassay kits are also envisionedcomprising a container having suitably aliquoted reagents for performingthe foregoing methods. For example, the containers may include one ormore bacterial cells with particular surface expressed analyte-bindingantibodies. Suitable containers might be vials made of plastic or glass,various tubes such as test tubes, metal cylinders, ceramic cups or thelike. Containers may be prepared with a wide range of suitable aliquots,depending on applications and on the scale of the preparation.Generally, this will be an amount in conveniently handled form, such asfreeze-dried preparations, and sufficient to allow rapid growth of thebacterial cells as required.

Such kits may optionally include surface-expressed antibodies in hostcells that are immobilized on surfaces appropriate for the intended use.One may for example, provide the cells attached to the surface ofmicrotiter plates, adsorbent resins, cellulose (e.g. filter paper),polymers, glass beads, etc.

F. Methods for Screening Libraries of Surface Displayed Polypeptides forthe Isolation of Polypeptides Capable of Binding to desired TargetMolecules

The present invention discloses general methods for the screening ofpolypeptide libraries for the isolation of polypeptides that recognizeand binding to desired target molecules with high affinity. Cellexpressing such high affinity polypeptides can then be readily employedfor immunoassays as described in section E. of the specification.

The screening of combinatorial libraries of polypeptides is greatlyfacilitated by the display of the polypeptides on the surface of hostcells. Simply, a cell population where each cell displays a differentpolypeptide is contacted with a desired ligand. The ligand is labeledsuch that it can allow the facile separation of cells that displaypolypeptides capable of binding the ligand. Examples of labels suitablefor the purposes of this invention include fluorescent dyes and magneticparticles. Alternatively the desired target molecule can be immobilizedon a suitable solid support. Cells producing surface displayedpolypeptides capable of binding the desired target molecule thus adhereto the immobilized support and can be readily separated from cells thatdo not bind to the immobilized support.

FACS Separation of Desired Cells Libraries of surface-displayedpolypeptides are rapidly and efficiently sorted using fluorescenceactivated cell sorting techniques (FACS). FACS permits the separation ofsubpopulations of cells initially on the basis of their light scatterproperties as they pass through a laser beam. Since cells are taggedwith fluorescent-labeled product, they can are characterized byfluorescence intensity and positive and negative windows set on the FACSto collect label⁺ (bright fluorescence) and label⁻ (low fluorescence)cells. Positive and negative windows are set to collect label⁺ andlabel⁻¹ cells, respectively. Cells are sorted at a flow rate of about3000 cells per second and collected in positive and negative cells.

Identification of antibody-expressing bacteria by FACS is directly basedon the affinity for the soluble hapten thus eliminating artifacts due tobinding on solid surfaces. This means only the high affinity antibodiesare recovered by sorting following binding of low concentrations offluorescently labeled antigen. There is no analogous method forspecifically selecting phage with very high affinity. Additionally, thesorting of positive clones is essentially quantitative. It is limitedonly by the accuracy of the flow cytometer, which is on the order of95%. In contrast with phage technology, the efficiency of selection isnot limited by avidity effects because screening does not depend onbinding to a surface having multiple antigens and thus the potential formultivalent attachment sites.

Magnetic Separation of Desired Cells E. Coli cells with surfaceexpressed polypeptides can be incubated with paramagnetic particles(e.g. Miltenyi Biotec, Bergisch Gladbach) that themselves are coatedwith an antigen of interest (Radbuch et al. 1994). Paramagneticparticles are available with a variety of surface derivatizationchemistries to allow for the covalent attachment of a wide range ofantigens. Bacteria having polypeptides with high affinity for theantigen remain bound to the magnetic particles, and the complexesisolated following washing steps in a strong magnetic field that retainsthe paramagnetic beads. Alternatively, cells labeled with paramagneticparticles can be separated in a continuous magnetic separator.

Separation of Cells by Adsorption onto Supports with Immobilized TargetMolecules In another embodiment of the present invention, cellsdisplaying polypeptides that bind to the desired target molecule may beisolated via selective adsorption onto solid matrices. In this case thecell population displaying the polypeptide library is contacted with asolid support in which the antigen is covalently immobilized viastandard chemical immobilization methodologies. Cells that displaypolypeptides capable of interacting with the immobilized targetmolecules are retained on the solid support and can be separated fromnon-binding cells. Following several washes with buffer to removenon-specifically adsorbed cells, the cells that are bound via specificinteractions are employed for further studies. Such specifically-boundcells can be dissociated from the solid support either by adding largeconcentrations of the soluble desired molecule to serve as a competitoror, alternatively by adding growth media to allow the cells to grow. Inthe latter case the progeny of the bound cells is released from thesolid support.

G. Methods for the Selection of Evolved Enzymes

The immobilization onto micron sized particles generally involvesbacteria being entrapped within agarose gel microdroplets (AGMs). Thismethod involves entrapping microorganisms in AGMs (10 to 100 microns indiameter) which are surrounded by a hydrophobic (low dielectric) fluid,subsequently distinguishing occupied and unoccupied AGMs withcolorimetric or fluorescence indicators, counting both occupied andunoccupied AGMs and applying statistical analyses to arrive atenumeration. It is possible to use a single preparation of AGMscontaining a range of AGM sizes, to simultaneously provide a viableenumeration of growing and non-growing cells.

The microdroplets are produced by first emulsifying a solutioncontaining bacteria and melted agarose. This results in the formation ofliquid microdroplets suspended in oil. By lowering the temperature atwhich the agarose is made to polymerize, thus forming micron sizeddroplets containing bacterial cells. AGMs of approximately 10 μM indiameter have been made using standard procedure. The conditions formaking AGMs that are occupied by one cell are well known to those ofskill in the art (Weaver et al., 1991).

This technique starts with a conventional cell suspension and generatesa large number (10⁶) of AGMs/ml by adding the cells to molten agaroseand dispersing into mineral oil. This suspension of AGMs is thentransiently cooled to a gelation state (Weaver et al., 1984; Weaver,1986; Williams et al., 1987; Weaver et al., 1988). Poisson statisticsallow the measurement of size of those AGMs that have a high probabilityof containing zero or one initial cell or colony forming units. The AGMscan be transferred out of the mineral oil into a suitable growth mediumand incubated to allow for the formation of microcolonies. These canthen be stained with fluorescent dyes for one or more generic indicatorsof biomass, for example, nucleic acids (stained with propidium iodide)or proteins (stained with FITC) and then measured using flow cytometrygenerally with single cell resolution. DNA staining may also be used andare well known to those of skill in the art (Bliss, 1990; Powell, 1989).

This assay method is applicable to any cell type which is amenable tobeing cultured in a gel-like matrix. The assay method has beensuccessfully demonstrated on mammalian, fungal and bacterial cells(Weaver et al., 1991). The method is sensitive to subpopulations withdifferent growth rates and may be used for working with a mixedpopulation of cells without the need for strain specific stains and isapplicable to any growth based assay.

For growth based assays the use of AGMs for the isolation of individualcells within AGMs thereby allowing both monoculture and mixed cellpopulations to be assayed. Further AGMs provide confinement of progenyof individual cells within AGMs, rapid measurement of large numbers ofindividual microcolonies by flow cytometry and a hybrid of plating andcell suspension culture. Other fundamental advantages include theextreme permeability of AGMs allowing convenient and rapid changes inculture conditions. AGMs respond rapidly to changes in physicalconditions (heat, electric field, light ionizing radiation), and theyare small and robust enough to be handled in a manner similar to thehandling of cells i.e. suspension, pipetting centrifuging and the like.It is possible to add additional matrix components (separately or incombination with agarose) to approximate a natural, complexextracellular environment and support the growth of cells which do notgrow in agarose alone (Scott, 1987; Nilsson et al., 1987; Akporiaye etal., 1988)

A variety of substrates can be used to assay the activity of, andultimately select for, desirable mutant enzymes out of surface-expressedmutant enzyme libraries. Here the gene for an enzyme to be mutated isexpressed on the surface of bacteria such as E. coli by cloning the genefor the enzyme into a surface-expression vehicle such as the LPP-OmpAsystem (Francisco et al., 1992, 1993). Mutant enzyme libraries can becreated from these gene constructs using known methods such as chemicalmutagenesis, error-prone PCR or amplification in a mutator strain.Identification of mutant enzymes with desirable properties such as novelsubstrate selectivity or remarkable catalytic activity can be achievedusing substrates that change an assayable property, i.e. fluorescenceintensity, ratio of multiple fluorophore emissions, antibody detectablestructural changes etc., upon catalytic action of the enzyme.

Numerous formats can be used to create the substrates with assayableproperties. For example, when assaying hydrolytic enzymes (lipases,esterases, phosphatases, etc.), a substrate can be synthesized that hasa fluorophore (fluorescein, bodipy, etc.) and a quencher (eosin,tetramethyl rhodamine, etc.) attached on either side of the hydrolyzedbond. Enzymatic cleavage will result in separation of the fluorophorefrom the quencher leading to an assayable increase in fluorophorefluorescence.

For an enzyme that synthesizes a single product from two or moresubstrates (ligases, aldolases, kinases, various biosynthetic enzymes),different fluorophores or chromophores can be attached to the differentindividual substrates. The ratio of the emissions can be determined, anda one-to-one ratio would indicate enzymatic reaction. Alternatively,only one substrate could contain the fluorophore, and the presence offluorophore emission on a product that is preferentially retained (seebelow) would indicate enzymatic reaction. Additional formats could beenvisioned in which quenchers are attached to one of the substrates anda fluorophore is attached to another, so that reaction leads toquenching of emission.

Finally, antibodies could be created that bind only products, notreactants, analogous to the detection used for cat-ELISA experiments(Tawfik et al. (1993)). Here the product-specific antibodies could belabeled with a fluorophore. The retention of the fluorophore emissionwould indicate the presence of product and hence an enzymatic reaction.This antibody labeling method should be applicable to almost anyenzymatic process, regardless of reaction type, as long an antibodiescould be found that can discriminate between substrates and products.

Since the mutant enzymes will be displayed on the surface of thebacteria, there is no impediment to diffusion of the assayable substrateto the mutant enzymes. This direct substrate access will allowsubstrates of virtually any size or shape to be used to assay forcatalytic activity, including polymeric species such as nucleic acids orpeptides, that would not reliably diffuse into cells.

An essential element of the experimental design is that thespectroscopically identifiable product must remain associated with thebacterium having the surface-expressed enzyme. This can be accomplishedby either of two methods.

In the first method, the product is electrostatically trapped on thebacterial surface. Bacteria such as E. Coli have negatively chargedsurfaces so that molecules with positive overall charge are retained onthe cellular surface. Substrate systems in which the product(s) of theenzymatic reactions posses substantially more positive charge thansubstrate(s) will have product preferentially retained on the bacteriumharboring the surface expressed mutant enzyme that carried out thereaction. By using low salt media, even after washing steps, the productcan be retained on the bacteria long enough for screening and selectionof the bacterial library using FACS. Here the gate of the FACS could beset to select for any number of parameters such as the presence of anunquenched fluorescent signal, the proper ratio between two fluorophoresof different emission wavelengths, the specific retention of a singlefluorophore, or the presence of product specific antibodies withcovalently attached fluorophores. One or several rounds of mutagenesisand/or FACS selection could be used for enrichment of bacteriaexpressing desired mutants, and the selected bacteria can be plateddirectly for screening of individual colonies, or regrown in liquidmedia in preparation for further rounds of FACS selection.

In the second method, the enzymatic reaction with substrate is carriedout in AGM's (Weaver et al., 1991) with enclosed bacteria from thesurface-expressed enzyme library. A solvent is chosen, such as mineraloil, to suspend the AGM's such that the product of the enzyme reactionis only soluble in the aqueous environment of the AGM surrounding thecell, not the mineral oil. This will ensure that the AGM with the mostactive enzyme will accumulate the most product. If the productfluorescence can be distinguished from substrate, i.e. via unquenching(hydrolysis of a fluorophore/quencher substrate) or quenching (synthesisof a product with both a fluorophore and a quencher from substrates thatcontained only one or the other) then the AGM's with desired enzymeactivities could be isolated by FACS or via fluorescence microscopyusing a micromanipulator.

For any of the previously mentioned selection strategies, the substratescan be changed dramatically all at once, or incrementally over thecourse of several rounds of selection. Either way, enzymes withdramatically different activities compared to wild type will beisolated.

I. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Surface Expression of Anti-digoxin Single Chain Fv Antibodies

E. coli strain JM109 [endA1 recA1 gyrA thi-1 hsdR17 (r_(k) ⁻ , M_(k)+)relA1 supE44 Δ(lac-proAB)/F′ traD36proAB lacl^(q) lacZΔM15] was used forall studies. pTX101 codes for an Lpp-OmpA-β-lactamase fusion (Franciscoet al., 1992). pTX152 codes for an Lpp-OmpA-scF_(v)(digoxin) fusion,where the scF_(v)(digoxin) is an anti-digoxin single chain F_(v)consisting of the heavy- and light-chain variable regions (V_(H) andV_(L)). The V_(H) and V_(L), joined by a 15 amino acid [(Gly)₄Ser]₃linker (Huston et al., 1988), were amplified from messenger RNA isolatedfrom two separate anti-digoxin hybridomas. An 11 amino acid peptide fromthe Herpes Simplex Virus glycoprotein (Novagen) was introduced at theC-terminus of the scF_(v) for analytical purposes. The presence of theHSV peptide allowed detection of the scF_(v)(digoxin) protein byreaction with a monoclonal antibody specific for the 11 amino acidepitope. The sequence of the single chain F_(v) antibody fragment isdisclosed in SEQ ID NO: 2. pTX152 was constructed by first removing thebla from pTX101 by digestion with EcoRI and BamHI. The amplified genecoding for the anti-digoxin scF_(v) was then digested with EcoRI andBamHI and ligated into pTX101. Both pTX101 and pTX152 carried thechloramphenicol resistance gene. Cultures were grown in LB medium(Difco) supplemented with 0.2% glucose and chloramphenicol (50 μg/ml) ata temperature of either 24° C. or 37° C.

Overnight cultures grown at 24° C. were harvested, resuspended in PBS atOD₆₀₀=2.0 and lysed by passage through a French pressure cell at 20,000psi. The lysates were then diluted with 1 volume of phosphate bufferedsaline containing 2.0% bovine serum albumin (PBS/2% BSA) and 5 mM of theprotease inhibitor phenylmethylsulfonyl fluoride (PMSF). 96 wellmicrotiter plates were incubated overnight at 37° C. with 100 μl of 100μg/ml of either bovine serum albumin (BAS) or digoxin-conjugated BSA(digoxin-BSA) in 0.1 M sodium carbonate buffer (pH 9.2). All subsequentsteps were carried out at room temperature. The wells were fixed for 5min with 100 μl methanol and were then blocked for 45 min with 200 μl ofpBS/1% BSA. After removing the blocking solution, the wells wereincubated for 2 hr with 100 μl of lysates, washed 3 times with 200 μlPBS/0.1% Tween 20 and incubated for 1 hr with 100 μl/well of monoclonalantibodies against the HSV peptide or antiserum against β-lactamase. Thewells were again washed 3 times with PBS/0.1% Tween, incubated for 1 hrwith 100 μl of the appropriate secondary antibodies conjugated withhorseradish peroxidase and were finally washed 5 times with PBS/0.1%Tween and 2 times with PBS. After addition of the substrate2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Pierce, Rockford,Ill.) the absorbance of each well was measured at 410 nm.

Whole cell ELISAs were performed as described above except that 100 μlsamples of overnight cultures that had been resuspended in PBS/1% BSA atOD₆₀₀=1.0 were used instead of cell lysates.

For fluorescence microscopy, overnight cultures grown at 24° C. wereharvested, resuspended at OD₆₀₀=0.5 in PBS containing 10⁻⁷ Mfluorescein-conjugated digoxin (digoxin-FITC; ILS LTD, London) and wereincubated at room temperature for 1 hr. Prior to microscopy the cellswere washed once with PBS and resuspended in equal volumes of PBS andVectashield mounting medium (Vector Laboratories) at an OD₆₀₀ ofapproximately 2.0.

The protein composition of whole cell membrane fractions isolated fromovernight cultures was analyzed by SDS-PAGE on 12% acrylamide gels andby Western blotting using anti-HSV monoclonal antibodies obtained fromNovagen Inc. (Madison, Wis.), and anti-OmpA antiserum. Whole membranefractions were prepared as described in Francisco et al. (1992).

Cells were grown in liquid culture at 37° C. and used to isolate totalmembranes. The presence of a band of the expected size (42 kDa) wasdetected in Western blots of whole cell membranes probed with antibodiesspecific for OmpA and for the HSV peptide (FIG. 1A). Cells containingpTX152 produced a protein which reacted with both the HSV-specific andthe OmpA specific sera, as expected for the Lpp-OmpA-scF_(v)(digoxin)fusion. Control cells containing pTX101 reacted only with OmpAantiserum. The lower molecular weight band in lanes 1 and 2 correspondsto the intact OmpA protein of E. coli.

The near absence of lower molecular weight bands crossreacting witheither the anti-HSV or the anti-OmpA antibodies indicated that thescF_(v)(digoxin) was not subjected to proteolysis ostensibly because itwas anchored on the cell surface and consequently is physicallyseparated from intracellular proteases. The intensity of theLpp-OmpA(46-159)-scF_(v)(digoxin) band in FIG. 1A is comparable to thatof the native OmpA band. The latter is a highly expressed protein thatis present in the E. coli outer membrane at about 100,000 copies percell (Lugtenberg & Van Alphen, 1983). The level of expressionLpp-OmpA(46-159)-scF_(v)(digoxin) is on the order of 50,000-100,000copies per cell.

The scF_(v) domain of the Lpp-OmpA-scF_(v)(digoxin) fusion proteinencoded by pTX152 was shown by ELISA to bind specifically to the haptendigoxin. Whole cell lysates from JM109/pTX152 and from JM109/pTX101 ascontrol were incubated on microtiter wells that had been coated witheither digoxin-conjugated BSA (digoxin-BSA) or unconjugated BSA.Subsequently, the wells were treated with antibodies against the HSVpeptide or β-lactamase to detect Lpp-OmpA(46-159)-scF_(v)(digoxin) orLpp-OmpA(46-159)-β-lactamase, respectively. FIG. 1A shows that lysatesfrom JM198/pTX152 bound specifically to wells coated with digoxin-BSAbut not to unconjugated BSA, whereas the lysates from the controlstrain, JM109/pTX101, did not give a signal with either. Thus,Lpp-OmpA(46-159)-scF_(v)(digoxin) is active and can bind to the haptenspecifically.

FIG. 1B shows the results of ELISAs using intact cells. Samplescontaining the same number of cells were used in all the studies. Cellscontaining the control plasmid, pTX101, gave the same low signal whenincubated on microtiter wells coated with either unconjugated BSA orwith digoxin-BSA. A similar weak signal was detected with JM109/pTX152incubated on BSA-coated wells and is presumably due to non-specificbinding. In contrast, a much higher absorbance was evident in wellscoated with the digoxin-BSA conjugate indicating that there are activefusion protein molecules on the cell surface.

The display of the active scF_(v) antibody on the cell surface wasconfirmed by fluorescence microscopy (FIG. 2B). JM109/pTX152 cells weregrown overnight at 24° C., incubated with a 1×10⁻⁷ M solution of adigoxin-FITC conjugate for 1 hour and washed. As shown in FIG. 2A andFIG. 2B, all of the cells visible with phase contrast microscopy gave astrong fluorescence signal. In control studies, when JM109/pTX101 cellswere incubated with the same concentration of digoxin-FITC and thenwashed, none of the cells became fluorescently labeled. Furthermore,protease treatment drastically reduced the ability of the cells to bindfluorescein-digoxin judging from the generation of a signal detectableby FACS.

The intensity of the fluorescence signal from JM109/pTX152 was dependenton the cell growth temperature and was much higher for cultures grown at24° C. instead of 37° C. This was consistent with previous resultsshowing that the amount of proteins expressed on the surface of E. coliby fusion to Lpp-OmpA(46-159) increased as the temperature is decreased(Francisco et al., 1992; 1993). Assuming that the efficiency of surfacedisplay in this case is similar to that of β-lactamase (Francisco etal., 1992), then at 24° C. virtually all the scF_(v) antibody chainsmust be accessible on the cell surface.

Samples of 10⁸ cells per ml from cultures grown at 24° C. were incubatedwith digoxin-FITC at 10⁻⁷ M, washed in buffer and diluted to 3×10⁶ cellsper ml prior to sorting. The samples were then analyzed using a FACSortflow cytometer. FIG. 3A and FIG. 3B show that the fluorescence intensityof JM101/pTX152 cells expressing a surface displayed recombinantantibody specific for digoxin was substantially higher than theintrinsic background signal of control E. coli cells. JM101/pTX101expressing Lpp-OmpA(46-159)-β-lactamase is used as a control.

When JM109/pTX152 cells were preincubated with an excess of free digoxinprior to incubation with the digoxin-FITC conjugate, the fluorescenceintensity of the cells was the same as for the background (FIG. 3D).This specific inhibition was also seen using fluorescence microscopy,and demonstrates that the surface expressed scF_(v)(digoxin)specifically binds the fluorescently labeled hapten in the binding siteand is not the result of nonspecific interactions.

Treatment of intact cells with trypsin prior to incubation withdigoxin-FITC almost completely eliminated the population offluorescently labeled cells detected by flow cytometry (FIG. 3C). Ingram-negative bacteria, the outer membrane serves as a barrier topreclude the diffusion of large extracellular molecules such asproteins. The action of trypsin is limited to the proteolysis ofproteins exposed on the external surface of E. coli (Kornacker &Pugsley, 1990). Thus, the above result provides further evidence thatthe active scFv(digoxin) is indeed accessible on the outer surface, freeto interact with molecules in solution.

The scF_(v)(digoxin) binding sites appeared to be fully saturated atconcentrations of digoxin-FITC above 10⁻⁷ M. Appreciable fluorescentsignal was clearly detected even at digoxin-FITC concentrations of 10⁻⁹M. These results are consistent with a binding constant that is at leastwithin an order of magnitude of the reported affinity of a solubleanti-digoxin scF_(v) antibody (Huston et al., 1988).

Example 2 Enrichment of Cells Displaying scF_(v) Antibodies by FACS

Antibody expressing cells were sorted essentially quantitatively from anexcess of control E. Coli in a single step. Specifically, in mixturescontaining JM109/pTX101 control cells at an excess of either 100:1 or1,000:1, the fraction of the total population that was sorted in thehigh fluorescence intensity window was 1.1% and 0.1% respectively (aftersubtracting the background), as expected from the ratio of input cells.

The use of FACS for isolating rare clones from a very large excess ofbackground was also demonstrated. JM109/pTX101 (cells displaying anunrelated protein on the cell surface) and JM101/pTX152 (cellsdisplaying the scFv(digoxin) antibodies) were mixed at a ratio of100,000:1 and labeled with digoxin-FITC. Following washing to remove anynon-specific binding of the digoxin-fluorescein conjugate on the controlE. Coli, 500,000 cells were run through the FACSort flow cytometer. Awide sorting gate, i.e., the minimum fluorescence required foracceptance of an individual cell, was selected such that up to 0.2% ofthe control cells fell within the sorting window. This ensured that allthe scF_(v)(digoxin) expressing cells would be recovered. The cellshaving an allowable fluorescence signal were collected and grown infresh media at 37° C.

An aliquot from that culture was used to inoculate fresh media andincubated at 24° C. Cells grown at this lower temperature were used forFACS because of the higher extent of surface display of thescF_(v)(digoxin) antibody on the surface of cells grown at 24° C.500,000 cells from this culture were run again through the FACSort andthose with a fluorescence within the allowable window were collectedautomatically under sterile conditions. The sorted population was grownfirst at 37° C. and then subcultures at 24° C. and was subjected to afinal round of sorting as above.

To ensure the complete absence of artifacts due to non-specific celladhesion in the flow path of the FACSort, each run was followed byextensive washing with bleach. FIG. 3E, FIG. 3F and FIG. 3G show thecell fluorescence distribution for the sorting runs. After only tworounds of growth and sorting, the fluorescence intensity of 79% of thecell population fell within the positive window. A similar enrichmentwas reproducibly obtained in three independent studies. These resultswere not due to a growth advantage of the cells expressingLpp-OmpA(46-159)-scF_(v)(digoxin), since successive regrowth of theinput cell mixture in the absence of sorting did not result in anydetectable enrichment.

To verify that the cells with the increased fluorescent signal after thefinal sorting step were indeed JM109/pTX152, a sample of cells from thefinal round of FACS was plated on chloramphenicol plates and thenreplica plated on plates containing 100 μg/ml ampicillin. The pTX152plasmid confers resistance only to chloramphenicol whereas the plasmidpTX101 which is present in the control cells also confers resistance toampicillin. Over 95% of all the colonies examined were chloramphenicolresistant and ampicillin sensitive (cm⁺, amp⁻), consistent with thephenotype expected for JM101/pTX152 cells. As an additional test,plasmid DNA was isolated from eight cm⁺, amp-colonies and the presenceof pTX152 was confirmed by restriction analysis.

The above results demonstrate that E. Coli displaying a recombinantantibody on its surface can be recovered from at least a 10⁵-fold excessof control E. coli simply by incubation with a fluorescent hapten andfluorescent activated cell sorting. Only two rounds of sorting andregrowth of the sorted cell population are needed for enrichment from alarge excess of background.

Example 3 Surface Expression of Anti-Digoxin Antibody IncorporatingProtease Cleavage Site

A construct similar to that used for surface expression of scF_(v)(digoxin) can be modified to incorporate a protease cleavage site. Forexample, the recognition sequence of enterokinase [(Asp)₄-Ile-Arg] canbe introduced in the Lpp-OmpA(46-159)-scF_(v) between the OmpA(46-159)and the scF_(v) domains. The protease cleavage site at the N-terminal ofthe scF_(v) antibody domain of the fusion protein is then used torelease the scF_(v) antibody in soluble form following treatment of thecells with the appropriate proteolytic enzyme. Because the outermembrane of E. coli serves as a protective barrier to the action ofexternally added proteases, very few contaminating proteins will bepresent in the culture supernatant. A single colony expressing a desiredsingle chain F_(v) antibody can be grown in liquid media and harvestedby centrifugation after overnight growth at 24° C. The cells areresuspended in buffer to maintain the pH approximately neutral. Proteaseadded at appropriate concentrations to the fusion protein to be treatedand incubated at least 4 hours at 4° C. will release the soluble singlechain F_(v). Subsequently the cell suspension is centrifuged and thesupernatant containing the solubilized single chain F_(v) antibody iscollected.

To separate the cleaved, soluble scF_(v) antibody from the protease, aswell as any E. coli trace contaminants, a His₆ sequence may beintroduced at the C-terminus of the scF_(v) using PCR amplification withthe appropriate primers. Polyhistidine tails bind strongly to metals sothat the fusion protein can be purified by immobilized metal-ionaffinity chromatography (IMAC). The separation of the cleaved scF_(v)antibody using is carried out as described by Georgiou et al. (1994).

Lpp-OmpA(46-159) fusions are typically expressed at a high level,typically around 5×10⁴ per cell. Thus the yield after protease treatmentand IMAC is at least 2-3 mg of antibody per liter of shake flaskculture.

Example 4 Solid Phase Immunoassays

This example illustrates a solid phase immunoassay using E. coli withanti-digoxin single chain F_(v) displayed on the surface. Thisimmunoassay is demonstrated to be a sensitive and quantitativetechnique. To facilitate the removal of the surface expressed antibodyfollowing the reaction with antigen, the cells may be attached to asolid support such as a membrane, dipstick or beads.

Digoxin-FITC (The Binding Site Inc. (San Diego, Calif.) was diluted to aconcentration of 20 nM in PBS. Digoxin (Sigma, St. Louis, Mo.) wasbrought to a concentration of 1 μM in PBS. The pTX152/JM109 cells weregrown in LB overnight at 37° C. then subcultured into fresh LB and grownovernight at room temperature. The cells were harvested and resuspendedin PBS pH 7.4 at a concentration of 10¹⁰ cells/ml, based on the O.D.₆₀₀to form a cell stock. Some cells were also resuspended in 15%glycerol/water and stored at 70° C. Frozen cells yielded the sameresults as freshly prepared cells.

The following reagents were transferred to a 1.5 ml microcentrifugetube, 25 μl of 20 nM digoxin-FITC solution, 0.5-50 μl of 1 μM digoxinand PBS to a final volume of 950 μl. The mixture was vortexed brieflyand pulsed in an Eppendorf microcentrifuge. A 25 μl or 100 μl aliquot ofthe cell stock (10¹⁰ cells/ml) was added to the mixture and allowed toincubate for 1 hour at room temperature. Following this incubation thecells were spun in a microcentrifuge for 5 minutes at 5,000 rpm and thesupernatant collected. The fluorescence of the digoxin-FITC in thesupernatant was measured using a fluorimeter.

This immunoassay was also performed with the cells attached to a solidsupport. The solid support was Fisher filter paper P5 was cut intostrips approximately 0.5 cm×2 cm, and dampened in PBS by submerging oneend of the filter paper and allowing it to move upward. The filter paperwas allowed to dry slightly and 10 μl of a solution containing 6×10⁹cells/ml was applied to the filter paper. The cells were fixed to thepaper with a solution of 6-PLP, 8% PFA and NaIO₄. The 6-PLP solution is10 mM 6-PLP, 200 mM MES, 700 mM NaCl, 50 mM KCl, 700 mM Lysine-HCl, 50mM MgCl₂, and 70 nM EGTA followed by 0.01M MgCl₂ and 8% PFA. The 8% PFAwas prepared by addition of 4 g of PFA into 50 ml of water, followed byheating the solution to 70° C. with constant stirring. Approximately 2drops of a 1M solution of NaOH was added to the solution until it becameclear. The solution was then filtered through Whatman filter paper #1,allowed to cool and stored at 4° C. until use. The final fixativesolution was produced by mixing the components A, B and C to a finalvolume of 10 ml just prior to use, where A is 1 ml 6-PLP, 04 g sucroseand 3.03 ml water; B is 21.4 mg NaIO₄; and C is 4.62 ml 8% PFA. Thefixative was applied dropwise to one end of the filter paper and allowedto soak upwards. The excess fixative was allowed to drain and the filterpaper was washed twice with a solution of 100 mM NH₄Cl. The filter papercan then be stored in a solution of PBS until use.

The assay was performed by removing the filter paper with the cellsattached and then measuring fluorescence of the solution.

Essentially quantitative assays for an antigen can be run using amixture of a known amount of labeled antigen combined with an unknownamount of unlabeled antigen. In one exemplary study, pTX152/JM109 withantidigoxin antibody displayed on the surface were grown in LB overnightat 37° C., subcultured into fresh LB and grown overnight at roomtemperature. The cells were pelleted and resuspended in PBS to form astock solution of cells at a concentration of 1×10¹⁰ cells/ml. A mixtureof 25 μl of 20 nm digoxin-FITC and an amount of 1 μM free digoxin in therange of 0.5-50 μl, was prepared and brought to a final volume of 950 μlwith PBS. A 25 μl or a 100 μl aliquot of the cell stock solution wasadded to the mixture to give a total of 0.25×10⁹ or 1×10⁹ cells. Themixture was then allowed to incubate at room temperature for 1 hour. Thecells were pelleted and the fluorescence of the supernatant was measuredfor each sample.

FIG. 4 shows a plot of the residual fluorescence observed with theindicated amount of free digoxin, 0.5 nM digoxin-FITC conjugate andeither 250 million or 1 billion cells expressing the antidigoxin singlechain antibody on their surface.

To facilitate the removal of the surface expressing antibody cellsfollowing the reaction, the cells may be attached to a solid support. Inthis example the solid support consisted of a membrane however manyother supports would work as well such as dipsticks or beads.

Strips of filter paper were moistened with PBS and allowed to dryslightly. A 10 μl aliquot of a 6×10⁹ cells/ml of a cell suspensioncontaining pTX152/JM109 cells displaying anti-digoxin antibodies ontheir surface was applied to the strips of pre-moistened filter paper.The cells were then fixed to the paper using a mixture of 6-PLP, PFA andNaIO₄, as described in the materials and methods, and washed twice witha solution of 100 mM NH₄Cl. Following the incubation no centrifugationis necessary the filter paper is simply removed from the solution andthe residual fluorescence measured as described previously.

The assays described in this example use fluorescence as the indicatorof binding; however, other indicator reactions may be used such asradioactivity, enzyme conjugates and when the surface expressed antibodyis catalytic, an assay for catalytic activity may be used.

Example 5 Discrimination of Surface Displayed Antibody Affinity by FlowCytometry

This example demonstrates that the display of antibodies on the surfaceof microorganisms constitutes the basis for discriminating betweenantibodies of different affinities. By adjusting the fluorescent antigenconcentration, it is possible to discriminate binders of high affinityfrom those with moderate affinity.

The heavy-chain residue Y33 of the scFv(digoxin) antibody is known to becritical for antigen binding (Short et al., 1995). The following mutantsof the scFv (digoxin) antibody: Y33N, Y33C, Y33S, Y33G, Y33STOP wereconstructed by overlap extension PCR™ (Ho, 1989). The large fragmentfrom EcoR I digested pSD192 was purified and religated to yield pSD195(AmpR). Primers Y33C.s, Y33N.s, Y33S.s Y33G.s, and Y33Stop.s along with3′ primer CMI.5 were used to amplify 3′ fragments for mutantconstructions. The 5′ overlap fragment was produced with primers #4 and#3 and overlapped with the mutant fragments using primers #4 and CMI.5.The resulting products were digested with EcoRI and ligated into pSDI95,and electroplated into JM109 to yield Cm resistant mutants pY33N, pY33C,pY33S, pY33G, and pY33STOP. Overnight cultures were grown at 37° C. andsubcultured 1:100 at 25 ° C. for 20 hrs. cells (200 ml) were harvestedinto 1 ml PBS pH 7.1-7.4, pelleted by centrifugation at 3000× g for 4min, and resuspended in 1.5 ml PBS. Cells were aliquoted into a cleaneppendorf tube and fluorescein-conjugated digoxin (The Binding Site,Eugene, Oreg.) was added to 1×10⁻⁷ M. Cells were incubated at roomtemperature (22-24° C.) for 1 hr with gentle shaking, pelleted andresuspended in I ml and analyzed by flow cytometry. At least 10,000events were acquired on a Becton Dickinson (San Jose, Calif.) FACSort.Parameters were set in LOG mode as follows: FSC threshold 80, FSC preampE01, FL1 800, SSC 400. Debris and other particulate material wereexcluded by defining an appropriate FSC-SSC around the cell population.

The relative affinity of the mutants constructed were, as determined byin vitro ELISA data, Y33N (moderate), Y33S (low), Y33G (background), andY33Stop (background). The flow cytometry data and previously obtainedELISA results (Burks et al., 1997), are shown in FIG. 5A and FIG. 5B,respectively. Importantly, the flow cytometry data for individual Y33mutants correlate with ELISA data. In particular, the mean fluorescencesignals vary in the manner WT>Y33N>Y33S>Y33G_Y33Stop. The resultsfurther demonstrate that the high affinity wild-type scFv may bediscriminated from the low affinity Y33S, and to a lesser extent, fromthe moderate affinity Y33N at concentrations of fluorescent-digoxinapproximately 20-fold greater than the dissociation constant.

Example 6 Flow Cytometric Assay for the Determination of Antigen BindingAffinities of Antibodies Displayed on Cell Surfaces

The availability of a fluorescent-antigen conjugate affords a simple andrapid method for flow cytometric affinity estimation of antibodiesdisplayed on the cell surface. In this example cell aliquots wereincubated with various concentrations of BODIPY-digoxin, diluted in PBS,and the mean total fluorescence was determined by flow cytometry. Fordetailed flow cytometric analysis, 20 ml were added directly from a 24hr, 25° C. culture to 180 ml BODIPY-digoxin at 10, 5, 2.5, 1. 25, 0.62and 0 nM in 96-well -plate, and allowed to incubate 1 hr with gentleshaking. Cell samples were diluted 1:5 in PBS for flow cytometricanalysis. Mean fluorescence was recorded for each sample, and theresulting data were used to plot a saturation curve and calculate therelative dissociation constant. Fluorescence histograms for thewild-type antibody expressing cells over a range of concentrations isshown in FIG. 6A. Normalized saturation curves for three mutants withapparent affinities within 1.5 fold of the wild type are shown in FIG.6B.

Example 7 Screening Antibody Libraries by Cell Surface Display andFluorescence-Activated Cell Sorting

Vector Construction for Surface Expression of scFv Libraries.

The successful construction and screening of polypeptide librariesdisplayed on the cell surface it was found to be important to constructnovel vectors that eliminate the probability of contamination of thelibrary with wild-type plasmid molecules.

Thus, pSD195 containing only the N-terminal portion of the cat geneimmediately downstream of the scFv to be expressed at the cell surface,was constructed. Two Pst I restriction sites were introduced, by silentmutagenesis, into the vector to simplify library construction. The firstsite was created just upstream of the scFv LCDR3 to be randomized andthe second was introduced within the chloramphenicol resistance gene,(CmR). Removal of the Pst I insert followed by religation generatedpSDI95, which does not allow host growth in the presence ofchloramphenicol (Cm), since cat is functionally inactivated. Thus, afterlibrary ligation and transformation, only clones that contain thecorrect PCR™ generated insert, in the correct orientation, will restoreCm resistance, and consequently, growth in the presence of Cm.

All DNA manipulations were as described elsewhere (Maniatis, 1996).Restriction enzymes were from Promega (Madison, Wis.), and Pfupolymerase from Stratagene (La Jolla, Calif.). Escherichia coli strainJM109 was used for all cloning steps and library experiments.Oligonucleotides for PCR™ reaction are shown in Table 4. Overnightcultures were grown at 37° C. in LB media supplemented with 0.2%glucose, ampicillin (100 μg/ml) and chloramphenicol (30 μg/ml), andsubcultured 1:100 at 25° C. for a specified duration. pTX152 wasdigested with Pvu I and BamH I, and the small fragment from pTX152 wasligated into similarly digested pET-22b to yield pGC183. Thechloramphenicol acetyl transferase gene (cat) was amplified from pBR325with primers CM.1.s and CM.2.as, digested with EcoR I and Sph I andligated into similarly digested pGC183 to yield pGC185. Finally, thedigoxin scFv was reintroduced into pGC185 by ligating the EcoR I-BamH Ifragment from pTX152 with similarly digested pGC185 to yield the pGC182(ampR, cmR). Subsequently, the Pst I site was removed from pGC182 byreplacing the AlwN I/Pvu I fragment with that from pUC18 giving rise topSD 182. Pst I sites were introduced upstream of the light-chain CDR3and within the cat gene by silent mutagenesis. Primers #4 and PIA.1as(Rxn1), PIA.2s and PIB.1as (Rxn2), and PIB.2s and SphI.2.as (Rxn3) wereused to amplify the corresponding fragments from pTX152, pSD182, andpBR322 respectively. Products from Rxn2 and Rxn3 were amplified withPIA.2 and Sph1.2as and the resulting product was overlapped with the RxnI product using primers #4 and SphI.2as, digested with EcoR I and Sph Iand ligated into similarly digested pSD182 to yield pSD192 (FIG. 7).TABLE 4 OLIGONUCLEOTIDE PRIMERS FOR LIBRARY CONSTRUCTION #4TGGACCAACAACATCGGT SEQ ID NO:3 CMI5 CCCATATCACCAGCTCACCGTCTTTC SEQ IDNO:4 CMI4 GACCCCGAGGACTAACGTCTTCGAATA SEQ ID NO:5 AATAC CM.1CCGAATTCGTTTGAACATGCCTAAC SEQ ID NO:6 CM.2 CGGAATTCGTGCGCAACACGATGAAGCSEQ ID NO:7 TC SPH1.2.AS AGGGCATGCAAGGGCACCAATAACTGC SEQ ID NO:8 CTTAP1A.1.AS TTGGCTGCAGTAATATATTGCAGCAT SEQ ID NO:9 P1A.2.STGCAATATATTACTGCAGCCAAACTAC SEQ ID NO:10 GCAT P1B.2.SCGGCAGTTTCTGCAGATATATTCGCAA SEQ ID NO:11 GAT P1B.1.ASCTTGCGAATATATCTGCAGAAACTGCC SEQ ID NO:12 GGAA P1B.ASACGCCACATCTTGCGAATATATCTGCA SEQ ID NO:13 GAAACTGCCGGAA #3CAGGGTACATTTTCACCG SEQ ID NO:14 LCDR3.S AACTGCAGCCAANNBACGCATNNBCCA SEQID NO:15 NNBACGTTCGGCTCGGGGA HCDR3.1.S GTATACTATTGCGCCGGCTCCTCTGGT SEQID NO:16 AACNNSNNSNNSNNSGATTATTGGGGT CATGGTGCT H2.ASGTTACCAGAGGAGCCGGCGCAATAGTA SEQ ID NO:17 TAC Y33N.STACATTTTCACCGACTTCAATATGAAT SEQ ID NO:18 TGGGTTCGC Y33C.STACATTTTCACCGACTTCTGCATGAAT SEQ ID NO:19 TGGGTTCGC Y33S.STACATTTTCACCGACTTCTCTATGAAT SEQ ID NO:20 TGGGTTCGC Y33G.STACATTTTCACCGACTTCGGGATGAAT SEQ ID NO:21 TGGGTTCGC Y33STOP.STACATTTTCACCGACTTCTAAATGAAT SEQ ID NO:22 TGGGTTCGC

Construction of Cell Surface Displayed scFv Libraries

The digoxin scFv light-chain codons for T91, V94, and P96 were chosenfor randomization. Both T91 and P96 form important contacts with digoxinin the Fab crystal structure (Jeffrey et al., 1993), suggesting thatthey play an important role in determining the antibody affinity.

Light-chain residues T91, V94 and P96 were randomized by PCR™ with Pfupolymerase (Stratagene, La Jolla, Calif.). Primers (Genosys, TheWoodlands, Tex.) LCDR.3.s and CMI.5 were used to amplify the LCDR3-cmR700bp fragment encoding the antibody LCDR3 and cat gene fragment withnon-Pst I containing pSD182 as a template to prevent wild-typecontamination. The resulting PCR™ product was digested with Pst I,purified by gel electrophoresis and electroelution and ligated into PstI digested, phosphatase treated pSDI95. The resulting plasmid librarywas electroporated into JM109 to yield 2×10⁵ transformants. After an 8hr growth in liquid media, light-chain library plasmid DNA was preparedto provide a stock for subsequent library screening experiments. Theprobability that each amino acid sequence is represented in the librarypool may be calculated, using a Poisson distribution (Lowman et al.,1991), to be greater than 85%. Flow cytometric analysis of ten randomlypicked clones showed 1/10 to weakly bind fluorescein-digoxin and 9/10clones exhibited no binding.

A second library was created by randomizing heavy-chain residues H99.K,H100.W, H100a.A, and H100b.M) using the NNS (S=G or C) randomizationscheme by overlap extension PCR (Ho, 1989). pSD182 was digested with XbaI and the large fragment was purified by gel electrophoresis, andreligated to yield pSD181. pSD181 was digested with Xba I and Apa I, andthe small fragment was gel purified and used as a template to producethe 3′ mutagenic antibody-cat fragment. The 5′ fragment was amplifiedfrom pTX152 with primers EcoRI.s and H3.as. After gel purification, 5′and 3′ fragments were subjected to overlap extension PCR with primers #4and PIB.as. PCR master mix (150 ml 10× Pfu buffer, 200 μM dNTPs, 8 ng/uLeach primer, 1 ng/uL 5′ and 3′ template fragments and Pfu (0.03 u/mL andwater to 1.5 ml) was transferred in 100 ml aliquots into thin-walleppendorf tubes and amplified as follows: 1 cycle 3 min (94° C.), 3.5min (49° C.). 3 min (76° C.), 30 cycles 2 min, 3.5 min, 3.5 min, 1 cycle7 min (76° C.). Products were combined, precipitated with ethanol,resuspended in 500 uL TrisHCl (10 mM) pH=8.5, and purified by gelelectrophoresis and electroelution. HC PCR DNA was digested with Pst I,precipitated, digested with Sal I, gel purified and electroeluted.pSD195 was digested with Sal I and Pst I Gel purified and electroeluted.Ligations (12 ug pSD195 Sal I/Pst I, 3.6 ug HC PCR Sal I/Pst I, 40 μlligase, 80 μl 10×, water to 800 μl) were incubated 20 hr at 16° C., heatinactivated 10 min at 70° C., ethanol precipitated, and resuspended in60 ul Tris-HCl. Four aliquots of 15 ul were individually electroporatedinto 300 ul electrocompetant JM109 cells. Electroporation cuvettes werewashed with 3 ml of SOC media, and incubated for 1 hr at 37° C. in atotal volume of 60 ml. Prewarned LB media (500 ml) supplemented with0.4% glucose, and 75 mg/ml ampicillin was added and 1:10 and 1:100dilutions were plated on LB plates supplemented with Cm (30 μg/ml) andAmp (100 μg/ml). Plating of dilutions revealed the library to contain4×10⁶ individual transformants. After 7 hrs of growth the culture wassubcultured 1:50 into 500 ml LB, Amp (100), Cm (30), 0.2% glucose, andgrown for 8 hr. Plasmid DNA was isolated to provide as a stock for HClibrary experiments. Flow cytometric analysis of the same twenty clonesshowed weak binding for three clones at 100 nM fluorescein-digoxin, and17/20 clones displayed levels of fluorescence consistent with backgroundor autofluorescence.

Clones that bind to the hapten digoxin were isolated from the library ina single step. This procedure is outlined in FIG. 8. Followingincubation of a library aliquot with fluorescein-digoxin, cellsdisplaying a total fluorescence greater than threshold value, which theuser may gradually decrease to reduce selection stringency, were sortedand recovered by vacuum filtration onto a 0.2 μm membrane (Millipore,Bedford, Mass.). The membrane was transferred to an agar platecontaining the appropriate antibiotics and incubated overnight at 37° C.Individual colonies were then grown in liquid media and assayed forhigh-affinity antigen binding by flow cytometry. Light-chain library DNA(3 μL) was transformed into JM109 by electroporation. Following a 12 hrsubculture at 25° C., 200 ul of the library culture was diluted into 1ml of sterile PBS, pelleted in an microcentrifuge at 3000 g for 5 min,and resuspended in 1.5 ml PBS. Library aliquots were incubated in thedark, with shaking, at 25° C. with fluorescein-digoxin tracer (10⁻⁷,10⁻⁸, 10⁻⁹ M) for 1 hr, pelleted and resuspended in PBS at approximately10⁸ cells/ml for flow cytometric analysis or sorting. 20,000 events wereacquired for each concentration (FIG. 9A-FIG. 9F). Highly fluorescentcells were asceptically sorted in exclusion mode and filtered onto a 0.2μm cellulose acetate membrane (Millipore, Bedford, Mass.). The filterwas transferred to an agar plate and incubated overnight at 37° C.Individual colonies were picked and grown at 37° C. to saturation,subcultured at 25° C. and grown 18 hr post selection analysis. Cellswere labeled as described above with fluorescent digoxin tracer (1×10⁻⁷M) and analyzed by flow cytometry. The amino acid sequences of clonesdisplaying high fluorescence were determined by DNA sequencing and theaffinity was characterized by flow cytometry. The combined results oftwo library sorting experiments are summarized in Table 5. TABLE 5 ScFv(dig) VARIANTS ISOLATED FROM A LIGHT-CHAIN LIBRARY BY FACS RelativeClone LCDR3 Fluorescence WT SQTTHVPPT SEQ ID NO:23 +++ LC1-1 SQATHMPGTSEQ ID NO:24 +++ LC1-2 SQTTHFPVT SEQ ID NO:25 +++ LC1-3 SQATHYPTT SEQ IDNO:26 +++ LC2-3 SQCTHWPVT SEQ ID NO:27 +++ LC2-4 SQTTHVPPT SEQ ID NO:28++ LC2-2 SQATHYPST SEQ ID NO:29 +++ LC2-6 SQATHSPST SEQ ID NO:30 +++PRESORT LC-1 SQVTHGPRT SEQ ID NO:31 + LC-2 SQGTHRPYT SEQ ID NO:32 + LC-3SQITHVPKT SEQ ID NO:33 + LC-4 SQLTHLPRT SEQ ID NO:34 LC-5 SQPTHVPPT SEQID NO:35 LC-6 SQVTHKPGT SEQ ID NO:36 − LC-7 SQLTHWPST SEQ ID NO:37 −LC-8* SQLTHGPRT SEQ ID NO:38 − LC-9* SQLTHGPRT SEQ ID NO:39 + LC-10SQZTHGPFT SEQ ID NO:40 −*LC-8 and LC-9 are unique at the DNA level+++ = 100 < mean fluorescence (10⁻⁷ M)++ = 60 < mean fluorescence < 100+ = 30 < mean fluorescence < 60− = < mean fluorescence < 30

The heavy-chain library was first screened in a single pass essentiallyas described for the light-chain library, to demonstrate that highaffinity scFv(dig) variants could be selected from large libraries usingonly a single FACS step. HCDR3 library DNA (3 ml) was electroporatedinto JM109 and grown 8 hr to saturation at 37° C. After a 14 hrsubculture at 25° C., 160 μl cells were harvested into 640 μl of 100,15, and 0 nM BODIPY-digoxin in PBS. Cells were incubated with gentleshaking for 45 min. Propidium iodide was added to a final concentrationof 5 μg/ml and cell were incubated 15 min. Cells were washed twice withPBS and resuspended to give a FACSort event rate of 1000 s⁻¹. Data from50,000 events was acquired for each concentration (FIG. 10A). Aftersterilization of the FACSort with 70% ethanol, a total of 10⁷ cells weresorted in recovery mode and 405 events were collected. Sorted cells werefiltered onto a 0.7 μm membrane (Millipore, Bedford, Mass.) andtransferred to a petri dish containing a sterile presoaked petri pad(Millipore, Bedford, Mass.) and allowed to incubate overnight at 37° C.192 colonies were picked with a pipette tip and grown in 200 μl in96-well plates overnight. Cells were subcultured 1:100 and grown at22-24° C. for 20 hr. In a fresh 96-well plates 10 μl cells was added to90μl of 18 nM BODIPY-digoxin. Cells were incubated 1 hr and diluted into500 ml PBS total for flow cytometric analysis. A total of 400 selectedclones were picked and grown in 96-well plates for a preliminary flowcytometric analysis. 95/190 exhibited mean fluorescence greater than100. Six clones did not grow. Plasmid DNA was purified from 40 clonesexhibiting the highest levels of fluorescence (mean fluorescenceintensity >150). Plasmid DNA was digested with Nco I to preventunnecessary analysis of clones possessing the wild-type restriction siteCGGATC. DNA sequencing results are shown in Table 6. TABLE 6 HEAVY-CHAINLIBRARY SCREENING RESULTS Clone HCDR3 K_(d)/K_(Dwt) WT SSGNKWAMDY SEQ IDNO:41 1 HC1.4 SSGNYRALDY SEQ ID NO:42 2.8 HC10.3 SSGNRRAWDY SEQ ID NO:431.3 HC10.2 SSGNRRALDY SEQ ID NO:44 1.2 HC8.1 SSGNGRAWDY SEQ ID NO:45 1.1HC1.3 SSGNISALDY SEQ ID NO:46 >10 HC2.1 SSGNQRKMDY SEQ ID NO:47 >5

TABLE 7 LCDR3 LIBRARY SPECIFICITY SORT SEQUENCES Clone LCDR3 RelativeFL1 WT SQTTHVPPT SEQ ID NO:48 +++ LC2-5 SQVTHRPLT SEQ ID NO:49 + LC2-7SQVTHDPGT SEQ ID NO:50 + LC3-1 SQVTHCPST SEQ ID NO:51 ++ LC3-7 SQVTHWPPTSEQ ID NO:52 +++ LC3-10 SQVTHYPVT SEQ ID NO:53 ++++ = 100 < mean fluorescence (10⁻⁷ M)++ = 60 < mean fluorescence < 100+ = 30 < mean fluorescence < 60− = < mean fluorescence < 30

The heavy-chain library was also screened using a multiple-step FACSprocess to enrich high-affinity antibody expressing cells. A libraryaliquot was labeled with BODIPY-digoxin at 70 nM, and sorted at 1200s⁻¹. Cells were sorted directly into supplemented LB media and grownwith shaking at 37° C. for 15 hrs. Cells were subcultured for 14 hrs andrelabeled with 5 nM BODIPY-digoxin. Cells were sorted a second time inexclusion mode and again amplified by growth. The final population wasrelabled with 5 nM BODIPY-digoxin and analyzed by flow cytometry.Fluorescence histograms for pre-enriched, as well as single anddouble-enriched populations are shown in FIG. 10B.

Selection for Altered Specificity

Selection based upon specificity was accomplished by preincubating thelight chain library with the digoxin analog, digitoxin, and thensubsequently labeling the preincubated cells with fluorescein-digoxin.An aliquot of the LCDR3 library was incubated with digitoxin (5×10⁻⁶ M)for 30 min at 25° C. with steady gentle inversion. Fluorescein-digoxinwas added to 10⁻⁷ M and cells were incubated an additional hour. Cellswere pelleted by centrifugation and resuspended to give a FACS eventrate of 1000 s⁻¹. A total of 10⁶ cells were screened and cellsdisplaying high fluorescence were collected by FACS. Colonies werepicked grown for FACS analysis. Plasmid DNA was prepared from clonesdisplaying a mean fluorescence greater than 50 for DNA sequencinganalysis. Sequencing analysis of five clones displaying highfluorescence showed a total consensus for the amino acid valine atwild-type position L91 (Table 5). Ten of ten clones selected in theabsence of digitoxin preincubation did not have a valine residue at thatposition.

Example 8 Isolation Of Cells Displaying Enzymes From A Vast Excess OfCells That Do Not Express An Active Enzyme

This example demonstrates that the enzymatic activity, rather than aligand binding activity, of a surface displayed polypeptide can be usedto isolate cells producing such a surface enzymatic activity from a vastexcess of cells that do not express enzymatically active polypeptides.The example specifically demonstrates how E. coli cells that express theprotease OmpT on their surface can be distinguished from cells that donot produce OmpT or produce inactive OmpT.

A substrate that becomes fluorescent upon cleavage by OmpT was designedby conjugating a fluorescent dye (BODIPY) and a quenching group(trimethylrhodamine) at the opposite ends of the secile bond. Enzymaticcleavage releases the quenching group into the medium resulting in theproduction of a fluorescent product. The fluorescent product wasdesigned to have several positive charges to allow its binding to thesurface of the cells via electrostatic interactions with the negativelycharged lipopolysaccharide molecules that comprises the outer layer ofthe E. coli surface. The chemical structure of the substrate is shown inFIG. 10.

The peptide moiety was synthesized at the University of Texas, Austinpeptide facility using standard FMOC coupling conditions. The thiol ofthe cysteine was alkylated with trimethylrhodamine iodoacetamide andthis product was acylated with BODIPY-FL succinamidyl ester (MolecularProbes). The crude product was purified by preparative HPLC.

Different strains of bacteria were exposed to the substrate for 10 min.and examined by FACS. The OmpT⁻ negative E. coli mutant UT5600, shows nofluorescence (FIG. 11A). However, UT5600 cells expressing OmpT from amulticopy plasmid (pML19), showed a much larger increase influorescence, which continued to increase for over 20 minutes. The meanfluorescence intensity of the OmpT⁺ cells was over 30 times higher thanthat of the cells without the plasmid (i.e., OmpT⁻ cells). Such adifference in OmpT fluorescence is more than sufficient to allow thesorting of cells expressing active enzyme from cells that do not expressOmpT (FIG. 11).

It was demonstrated that inactive OmpT mutants exhibit increasedfluorescence upon incubation with the substrate. For this purpose, anOmpT mutant was produced in which the conserved His212 residue (which isthought to be part of the catalytic triad) was converted to Ala bysite-directed mutagenesis (Maniatis et al. 1989). The His212->Ala mutantwas confirmed to have no OmpT activity. UT5600 cells expressing theHis212->Ala produce the same amount of OmpT as cells transformed with aplasmid encoding the wild type enzyme. When UT5600 cells expressing theinactive OmpT mutant were incubated with the substrate and examined byFACS they exhibited a background fluorescence that could be clearlydistinguished from that of OmpT positive cells.

In other studies it was demonstrated that OmpT⁺ cells can be readilyisolated from a population containing a huge excess of OmpT⁻ cells.Specifically, OmpT⁺ cells were mixed with OmpT⁻ cells at a 5,000-foldexcess. The cell mixture was incubated with the substrate, passedthrough the fluorescence activated cell sorter and cells exhibiting ahigh fluorescence intensity were isolated. Nine out of nine sortedclones that were isolated produce OmpT. These studies showed that theOmpT⁺ cells can be readily isolated by FACS from a huge excess ofbackground cells solely on the basis of the enzymatic activity of theOmpT protease (FIG. 12).

Example 9 Isolation Of Clones Expressing Desired Enzymes FromCombinatorial Polypeptide Libraries Displayed On The Cell Surface

This example illustrates how libraries of polypeptides displayed on thecell surface can be screened to isolate clones that produce polypeptideshaving a desired enzymatic activity. Specifically this example teachesthe screening of libraries of OmpT mutant polypeptides to isolate novelenzymes that can hydrolyze peptide sequences not recognized by thewild-type OmpT enzyme.

The ompT gene will first be subjected to random mutagenesis usingestablished techniques such as error-prone PCR, chemical mutagenesis ormutator strains. For error-prone PCR or chemical mutagenesis the ompTgene is first excised from the high copy plasmid pML19 by digestion withappropriate restriction endonucleases and mutagenized according tostandard procedures known to those skilled in the art (Maniatis et al.1989, Innis et al. 1990). Following mutagenesis, the ompT DNA will beligated back to restriction-enzyme digested pML19 and the ligationmixture will be electroporated into E. Coli UT5600. Transformants willbe grown in LB broth containing ampicillin (100 μg/ml) and glucose at0.2% w/v at 37° C. Cultures will be grown to saturation to ensuremaximal expression of OmpT. Subsequently, the cells will be harvested bycentrifugation, washed with PBS and resuspended in 1 mM Tris buffer, pH7.0, in the presence of a substrate having a structure similar to theone shown in FIG. 10 except that the Arg-Arg dipetide that is recognizedby the wild type OmpT is substituted with other dipeptide sequences, forexample Arg-His, Arg-Ala, His-His, etc. Cells expressing OmpT capable ofhydrolyzing the substrate allow the release of the trimethylrhodaminequencher into the solution while the N-terminal cleavage productcontaining the BODIPY fluorophore is electrostatically retained by thecells. As a result, cells displaying mutant OmpT proteins capable ofhydrolyzing the substrate will become fluorescent and can thus beisolated by fluorescent activated cell sorting (FACS). For the isolationof enzymatically active cells by FACS, a gate is set such that onlycells exhibiting high fluorescence are sorted in the positive window.Cells will be sorted at an event rate of at least 1000 s⁻¹. A total of10⁶ cells will be screened and cells displaying high fluorescence willbe collected by FACS. Isolated colonies will be then screened forproduct hydrolysis by FACS analysis. Finally, the DNA sequence of themutant ompT genes encoding enzymes with altered substrate specificitywill be determined by DNA sequencing.

All of the composition and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

J. References

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Agterberg, et al., “Outer-membrane PhoE protein of Escherichia coli    K-12 as an exposure vector: possibilities and limitations”, Gene    88:37-45, 1990-   Aiyar & Leis, “Modification of the Megaprimer Method of PCR    Mutagenesis: Improved Amplification of the Final Product,”    BioTechniques, 14:366-369, 1993-   Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988-   Baneyx and Georgiou, J. Bacteriol., 172:491-494, 1990.-   Barbas et al., “Assembly of combinatorial antibody libraries on    phage surfaces: The gene III site,” Proc. Natl. Acad. Sci. USA,    88:7978-7982, 1991-   Barbas et al., “Semisynthetic combinatorial antibody libraries: A    chemical solution to the diversity problem,” Proc. Natl. Acad. Sci.    USA, 89:4457-4461, 1992-   Better et al., “Escherichia coli Secretion of an Active Chimeric    Antibody Fragment,” Science, 240:1041-1043, 1988-   Blackburn et al., “Electrochemiluminescence Detection for    Development of Immunoassays and DNA Probe Assays for Clinical    Diagnostics,” Clinical Chemistry, 37(9):1534-1539, 1991-   Boder, E. T. and Wittrup, K. D., “Surface display of a functional    single chain Fv antibody in Saccharomyces cerevisiae” Submitted 1996-   Brennan, Chemical and Eng. News, 74:31033, 1996-   Brown, S., “Engineered iron oxide-adhesion mutants of the    Escherichia coli phage λ receptor”, Proc. Natl. Acad. Sci. USA    89:8651-8655, 1992-   Burks, E. A., Chen, G., Georgiou, G., Iverson, B. L. “In vitro    Scanning Saturation Mutagenesis of an Antibody Binding Pocket,”    Proc. Natl. Acad. Sci. 94:412-7 (1997).-   Cadwell and Joyce, PCR Meth. Appl.:2, 28, 1992-   Campbell, in: Monoclonal Antibody Technology Laboratory Techniques    in Biochemistry and Molecular Biology, Vol. 13, Burden & Von    Knippenberg, Amsterdam, Elseview, pp. 75-83, 1984-   Charbit, et al., “Versatility of a vector for expressing foreign    polypeptides at the surface of Gram-negative bacteria”, Gene    70:181-189, 1988-   Chiswell & Clackson, 1992-   Chiswell & McCafferty, “Phage antibodies: will new ‘coliclonal’    antibodies replace monoclonal antibodies?” TIBTECH, 10:80-84, 1992-   Chou & Fasman, “Conformational Parameters for Amino Acids in    Helical, β-Sheet, and Random Coil Regions Calculated from Proteins,”    Biochemistry, 13(2):211-222, 1974b-   Chou & Fasman, “Empirical Predictions of Protein Conformation,” Ann.    Rev. Biochem., 47:251-276, 1978b-   Chou & Fasman, “Prediction of β-Turns,” Biophys. J., 26:367-384.    1979-   Chou & Fasman, “Prediction of protein conformation,” Biochemistry,    13(2):222-245, 1974a-   Chou & Fasman, “Prediction of the Secondary Structure of Proteins    from Their Amino Acid Sequence,” Adv. Enzymol. Relat. Areas Mol.    Biol., 47:45-148, 1978a-   Clackson et al., “Making antibody fragments using phage display    libraries,” Nature, 352:624-628, 1991-   Cornellis, et al., “Development of new cloning vectors for the    production of immunogenic outer membrane fusion proteins in    Escherichia coli.”, Bio/Technology 14:203-208, 1996-   Fischetti, V. A., “Gram-positive commensal bacteria deliver antigens    to elicit mucosal and systemic immunity”, ASM News 62:405-410, 1996-   Francisco et al., “Production and fluorescence-activated cell    sorting of Escherichia coli expressing a functional antibody    fragment on the external surface,” Proc. Natl. Acad. Sci. USA,    90:10444-10448, 1993-   Francisco et al., Proc. Natl. Acad. Sci. USA, 89:2713-2717, 1992-   Francisco, Earhart, Georgiou, Proc. Nat'l Acad. Sci. USA,    89:2713-2718, 1992-   Francisco, et al., “Transport and anchoring of β-lactamase to the    external surface of Escherichia coli.”, Proc. Natl. Acad. Sci. USA    89:2713-2717, 1992.-   Francisco, et al., Bio/Technology, 11:491-496, 1993-   Frohman, In: PCR Protocols: A Guide to Methods and Applications,    Academic Press, N.Y., 1990-   Fuchs, et al., “Targeting recombinant antibodies to the surface of    Escherichia coli: fusion to a peptido-glycan-associated    lipoprotein”, Bio/Technology 9:1369-1372, 1991-   Gefter et al., Somatic Cell Genet., 3:231-236, 1977-   Georgiou, et al., “Display of heterologous Molecules on the Surface    of Microorganisms: From the Screening of Combinatorial Libraries to    Live Bacterial Vaccines,” Nature Biotechnol. 15:29-35 1997.-   Georgiou, et al, “Practical applications of engineering    Gram-negative bacterial cell surfaces”, Trends Biotechnol. 11:6-10,    1993.-   Georgiou, G. et al, “folding and Aggregation of TEM β-lactamase:    Analogies with the Formation of Inclusion Bodies in Escherichia    coli”, Protein Science, 3:1953-60, 1994.-   Georgiou, Stephens, Stathopoulos, Poetschle, Mendenhall, Earhart,    Protein Engineer., 9:239-247, 1996.-   Goding, in: Monoclonal Antibodies: Principles and Practice, 2nd Ed.,    Orlando, Fla., Academic Press pp. 60-61, 65-66, 71-74, 1986-   Hansson, et al., “Expression of recombinant proteins on the surface    of the coagulase-negative bacterium Staphylococcus xylosus ”, J.    Bacteriol. 174:4239-4245, 1992-   Harlow & Lane (Eds.), Antibodies: A Laboratory Manual, Cold Spring    Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988-   Helmann and Chamberlain, “Structure and function of bacterial sigma    factors.” Ann. Rev. Biochem. 57: 839-872, 1988.-   Ho et al., 1989-   Hofnung, M., “Expression of foreign polypeptides at the Escherichia    coli cell surface”, Methods Cell Biol. 34:77-105, 1991-   Hollfelder, Kirby, Tawfik, Nature, 383:60-63, 1996-   Huse et al., “Generation of A Large Combinatorial Library of the    Immunoglobulin Repertoire in Phage Lambda,” Science, 246:1275-1281,    1989-   Huston et al., 1989-   Huston et al., Methods in Enzymology, “Molecular Design and    Modeling: Concepts and Applications,” Abelson & Simon, Academic    Press, Inc., San Diego, pp. 46-99, 1991-   Huston, et. al., “Protein engineering of antibody binding sites:    Recovery of specific activity in an anti-digoxin single-chain Fv    analogue produced in Escherichia coli,” Proc. Natl. Acad. Sci. USA,    85:5879-5883, 1988-   Innis et al., PCR Protocols, Academic Press, Inc., San Diego Calif.,    1990-   Iverson et al., “A Combinatorial System for Cloning and Expressing    the Catalytic Antibody Repertoire in Escherichia coli,” Cold Spring    Harbor Symposia on Quantitative Biology, 54:273-281, 1989-   Jameson & Wolf, “The Antigenic Index: A Novel Algorithm for    Predicting Antigenic Determinants,” Comput. Appl. Biosci.,    4(1):181-186, 1988-   Janda, et al., J. Am. Chem. Soc., 110:835-4837, 1988-   Jeffrey, P. D., Strong, R. K., Sieker, L. C., Chang, C. Y.,    Campbell, R. L., Petsko, G. A., Haber, E., Margolies, M. N. &    Sheriff, S. 26-10 Fab-digoxin complex: affinity and specificity due    to surface complementarity. Proc. Nat'l Acad. of Sci. 90, 10310-4    1993.-   Jose, et al., “Common structural features of IgA1 protease-like    outer membrane protein”, Mol. Microbiol. 18:378-380, 1995-   Kabat et al., Sequences of Proteins of Immunological Interest, U.S.    Dept. of Health and Human Services, U.S. Government Printing Office,    1987-   Kang et al., “Linkage of recognition and replication functions by    assembling combinatorial antibody Fab libraries along phage    surfaces,” Proc. Natl. Acad. Sci. USA, 88:4363-4366, 1991-   Klauser, et al., “Extracellular transport of cholera toxin B subunit    using Neisseria IgA protease β-domain: conformation-dependent outer    membrane translocation”, EMBO J. 9:1991-1999, 1990-   Klauser, et al., “The secretion pathway of IgA protease-type    proteins in Gram-negative bacteria”, Bioessays 15:799-805, 1993-   Kohler & Milstein, Eur. J Immunol., 6:511-519, 1976-   Kohler & Milstein, Nature, 256:495-497, 1975-   Kornacker & Pugsley, Mol. Microbiol, 4:1101-1109, 1990-   Kornacker, et al., “The normally periplasmic enzyme β-lactamase is    specifically and efficiently translocated through the Escherichia    coli outer membrane when it is fused to the cell-surface enzyme    pullulanase”, Mol. Microbiol. 4:1101-1109, 1990-   Kuby, J. “Immunology”, W.H. Freeman and Co., New York (1990)-   Kwoh et al., “Transcription-based amplification system and detection    of amplified human immunodeficiency virus type I with a bead-based    sandwich hybridization format,” Proc. Nat. Acad. Sci. USA,    86:1173-1177, 1989-   Laukkanen, et al., “Lipid-tagged antibodies: bacterial expression    and characterization of a lipoprotein-single-chain antibody fusion    protein”, Protein Engineer 6:449-454, 1993-   Lerner, Benkovic, Schultz, Science, 252:659-667, 1991-   Liad, MvKenzie, Hageman, Proc. Nat'l Acad. Sci. USA, 83:576-580,    1986-   Lilley and Higgins, “Local DNA topology and gene expression: the    case of the leu-500 promoter.” Mol. Microbiol. 5, 779-783.-   Lu, et al., “Expression of thioredoxin random peptide libraries on    the Escherichia coli cell surface as functional fusions to    flagellin: A system designed for exploring protein-protein    interactions”, Bio/Technology 13:366-372, 1995-   Lugtenberg & Van Alphen, “Molecular architecture and functioning of    the outer membrane of Escherichia coli and other gram-negative    bacteria,” Biochem. Biophys. Acta, 737:51-115, 1983-   Makrides S C, Microbiol. Rev. Vol. 60(3):512-538, 1996.-   Mangel, Toledo, Brown, Worzalla, Lee, Dunn, Methods Enzymol,    244:384-399, 1994-   Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold    Springs Harbor Laboratories, Cold Springs Harbor, N.Y., 1982-   Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold    Springs Harbor Laboratories, Cold Springs Harbor, N.Y., 1989-   Matthews, Adv. Prot. Chem., 46:249-278, 1995.-   McCafferty et al., “Phage antibodies: filamentous phage displaying    antibody variable domains,” Nature, 348:552-554, 1990-   Moore and Arnold, Nature Biotechnol., 14:458-467, 1996-   Morrison & Desrosiers, “A PCR-Based Strategy for Extensive    Mutagenesis of a Target DNA Sequence,” BioTechniques, 14:454-457,    1993-   Mullinax et al., “Identification of a human antibody fragment clones    specific for tetanus toxoid in a bacteriophage λ immunoexpression    library,” Proc. Natl. Acad. Sci. USA, 87:8095-8099, 1990-   Nakamura et al., Enzyme Immunoassays: Heterogeneous and Homogeneous    Systems, Ch. 27, 1987-   Newton, et al., “Expression and immunogenicity of an 18-residue    epitope of HIVI gp41 inserted in the flagellar protein of a    Salmonella live vaccine, Res. Microbiol. 146: 203-216-   Newton, et al., “Topology of the membrane protein LamB by epitope    tagging and a comparison with the X-ray model”, J. Bacteriol.    178:3447-3456, 1996-   O'Callaghan, et al., “Immunogenicity of foreign peptide epitopes    expressed in bacterial envelope proteins”, Res. Microbiol.    141:963-969, 1990-   O'Hara, et al., “One-sided polymerase chain reaction: The    amplification of cDNA,” Proc. Nat'l Acad. Sci. USA, 86:5673-5677,    1989-   Orlandi et al., “Cloning immunoglobulin variable domains for    expression by the polymerase chain reaction,” Proc. Nat. Acad. Sci.    USA, 86:3833-3837, 1989-   Osuna, Flores, Sobeon, Crit. Rev. Microbiol., 20:107-116, 1994-   Pallesen, et. al., “Chimeric FimH adhesin of type I fimbriae: a    bacterial surface display system for heterologous sequences”,    Microbology 141:2839-2848, 1995-   Palzkill et al., “Selection of functional signal peptide cleavage    sites from a library of random sequences.” J Bacteriol, 176 (3)    p563-8, 1994-   Parmley & Smith, “Antibody-selectable filamentous fd phage vectors:    affinity purification of target genes,” Gene, 73:305-318, 1988-   Persson et al., “Generation of diverse high-affinity human    monoclonal antibodies by repertoire cloning,” Proc. Natl. Acad. Sci.    USA, 88:2432-2436, 1991-   Petrosino and Palzkill, J. Bacteriol., 178:1821-1828, 1996-   Pozzi, et al., “Delivery and expression of a heterologous antigen on    the surface of streptococci”, Infect. Immun. 60:1902-1907, 1992-   Provence, et al., “Analysis of the extracellular secretion of the    putative hemagglutinin Tsh of an avian pathogenic Escherchia coli    strain: evidence for an autonomous IgA protease-type secretion”, In    Preparation, 1997-   Salmong, G. P. C. and Reeves, P. J., “Membrane traffic wardens and    protein secretion in Gram-negative bacteria”, Trends Biol. Sci.    18:7-12, 1993-   Samuelson, et al., “Cell surface display of recombinant proteins on    Staphylococcus carnosus”, J. Bacteriol. 177:1470-1476, 1995-   Sastry et al., “Cloning of the immunological repertoire in    Escherichia coli for generation of monoclonal catalytic antibodies:    Construction of a heavy chain variable region-specific cDNA    library,” Proc. Natl. Acad. Sci. USA, 86:5728-5732, 1989-   Schildbach et al., Protein Science, 2:206-214, 1993-   Schreuder, et al., “Immobilizing proteins on the surface of yeast    cells”, Trends Biotechnol. 14:115-120, 1996-   Schreuder, et al., “Yeast expressing hepatitis B virus surface    antigen determinants on its surface: implications for a possible    oral vaccine”, Vaccine 14:383-388, 1996-   Schreuder, et. al., “Targeting of a heterologous protein to the cell    wall of Saccharomyces cerevisiae ”, Yeast 9:399-409, 1993-   Scott & Smith, “Searching for Peptide Ligands with an Epitope    Library,” Science, 249:386-390, 1990-   Skerra & Plückthun, “Assembly of a Functional Immunoglobulin F_(v)    Fragment in Escherichia coli,” Science, 240:1038-1041, 1988-   Smiley and Benkovic, “Selection of catalytic antibodies for a    biosynthetic reaction from a combinatorial cDNA library by    complementation of an auxotrophic Escherichia coli: antibodies for    orotate decarboxylation.” Proc Nat'l Acad Sci., 91 (18) p 8319-23,    1994.-   Smith “Surface presentation of protein epitopes using bacteriophage    expression systems.” Curr. Opin. Biotechnol. 2 (5) p 668-73, 1991.,    1991-   Smith et al., 1992-   Sousa, et al., “Enhanced metalloadsorption of bacterial cells    displaying poly-His peptides”, Nature Biotech. 14:1017-1020, 1996-   St. Geme, et al., “A Haemophilus influenzae IgA protease-like    protein promotes intimate interaction with human epithelial cells”,    Mol. Microbiol. 14:217-233, 1994-   Stathopoulos et al., Appl. Microbiol. Biotechnol. 45:112-119, 1996-   Stathopoulos, et al., “Characterization of Escherichia coli    expressing an Lpp'OmpA(46-159)-PhoA fusion protein localized in the    outer membrane”, Appl. Microbiol. Biotechnol. 45:112-119, 1996-   Steidler, et al., “Pap pili as a vector system for surface for    surface exposition of an immunoglobulin G-binding domain of protein    A of Staphylococcus aureus in Escherichia coli. ”, J. Bacteriol.    175:7639-7643, 1993-   Stemmer, Nature, 370:389-391, 1994-   Stemmer, Proc. Nat'l Acad. Sci. USA, 91:10747-10751, 1994-   Su, et al., “Construction of stable LamB-Shiga toxin B subunit    hybrids: analysis of expression in Salmonella typhimurium aroA    strains and stimulation of B subunit-specific mucosal and serum    antibody responses”, Infect. Immun. 60:3345-3359, 1992-   Suzuki, et al., “Extracellular transport of VirG protein in    Shigella”, J. Biol. Chem. 270:30874-30880, 1995-   Tang, Hicks, Hilvert, Proc. Nat'l Acad. Sci. USA, 88:8784-8786, 1991-   Tanke & van der Keur, Trends Biotechnol., 11:55-62, 1992-   Tawfik, Green, Chap, Sela, Eshhar, Proc. Nat'l Acad. Sci. USA,    90:373-377, 1993-   Taylor, et al., “The TraT lipoprotein as a vehicle for the transport    of foreign antigenic determinants to the cell surface of Escherichia    coli K12: structure-function relationship in the TraT protein”, Mol.    Microbiol. 4:1259-1268, 1990-   van Die, et al., “Expression of foreign epitopes in P-fimbriae of    Escherichia coli.”, Mol. Gen. Genet. 222:297-303, 1990-   Venkatachalam, Huang, Larocco, Palzkill, J. Biol. Chem.,    269:23444-23450, 1994-   Walker et al., “Isothermal in vitro amplification of DNA by a    restriction enzyme/DNA polymerase system,” Proc. Nat'l Acad. Sci.    USA, 89:392-396, 1992-   White, Chen, Kenyon, Babbitt, J. Biol. Chem., 270:12990-12994, 1995-   Winter & Milstein, “Man-made antibodies,” Nature, 349:293-299, 1991-   Wolf et al., “An Integrated Family of Amino Acid Sequence Analysis    Programs,” Comput. Appl. Biosci., 4(1):187-191, 1988-   Wong, et al., “Pseudomonas aeruginosa outer membrane protein OprF as    an expression vector for foreign epitopes: the effects of    positioning and length on the antigenicity of the epitope”, Gene    158:55-60, 1995-   Wu et al., “The Ligation Amplification Reaction (LAR)—Amplification    of Specific DNA Sequences Using Sequential Rounds of    Template-Dependent Ligation,” Genomics, 4:560-569, 1989-   Yang et al., “Electrochemiluminescence: A New Diagnostic and    Research Tool,” Bio/Technology, 12:193-194, 1994-   Yu and Arnold, Protein. Eng., 9:77-83, 1996.

1. A method for selecting a polypeptide from a plurality of candidateproteins, the method comprising the steps of: (a) obtaining a library ofvectors that encode a plurality of distinct candidate polypeptides,wherein said vector provides for the cell surface expression of saidcandidate polypeptides; (b) expressing each of said plurality ofcandidate polypeptides on the surface of a host cell; and (c) selectinga host cell that expresses a desired polypeptide.
 2. The method of claim1, wherein said host cell is a Gram negative bacterium.
 3. The method ofclaim 2, wherein said host cell is E. coli.
 4. The method of claim 1,wherein said polypeptide is selected from the group consisting of anantibody or antibody fragment, an enzyme, a cytokine, a transcriptionfactor, a clotting factor, a chelating agent, a hormone and a receptor.5. The method of claim 4, wherein said polypeptide is an antibody orantibody fragment.
 6. The method of claim 5, wherein selecting a hostcell that expresses a desired antibody comprises the steps of: (a)contacting said antibody- or antibody fragment-expressing cells with aselected antigen; and (b) identifying a host cell that binds to saidselected antigen.
 7. The method of claim 6, wherein the antigen islabeled.
 8. The method of claim 7, wherein the label is a fluorescent orchemilluminescent label.
 9. The method of claim 6, wherein said selectedantigen is located on the surface of a cell other than said host cell,and said host cell that binds to said selected antigen is identified bya method comprising the steps of: (a) contacting said host cell withsaid cell expressing or having conjugated thereto said selected antigen;and (b) identifying a host cell bound to said cell expressing or havingconjugate thereto said selected antigen.
 10. The method of claim 9,further comprising size sorting of bound cells following the step ofcontacting said host cell with said cell expressing or having conjugatedthereto said selected antigen.
 11. The method of claim 6, wherein saidvector library is obtained by a method comprising the steps of: (a)administering to an animal an immunologically effective amount of acomposition comprising a selected antigen; (b) obtaining from the animala plurality of distinct DNA segments that encode distinct antibodies orantibody fragments; and (c) incorporating said plurality of DNA segmentsinto a plurality of expression vectors, the vectors expressingantibodies or antibody fragments on the outer membrane surface of a Gramnegative host cell.
 12. The method of claim 11, wherein said pluralityof DNA segments are obtained by a method comprising the steps of: (a)isolating mRNA from antibody-producing cells of said animal; (b)amplifying a plurality of distinct RNA segments using a set of nucleicacid primers having sequences complementary to antibody constant regionor antibody framework region nucleic acid sequences; and (c) preparing aplurality of distinct DNA segments having sequences complementary tosaid amplified RNA segments.
 13. The method of claim 1, wherein saidvector library is obtained by a method comprising the steps of: (a)obtaining a DNA segment that encodes a selected polypeptide; (b)mutagenizing said DNA segment to provide a plurality of DNA segmentsthat encode a plurality of polypeptides; and (c) incorporating saidplurality of DNA segments into a plurality of expression vectors, thevectors expressing a plurality of polypeptides on the surface of a Gramnegative host cell.
 14. The method of claim 13, wherein said polypeptideis an antibody or an antibody fragment.
 15. The method of claim 14,wherein said selected cells that express a desired antibody aresubjected to cleavage to release the selected antibody or antibodyfragment from the surface of the outer membrane.
 16. The method of claim13, wherein selecting a host cell that expresses a desired antibodycomprises the steps of: (a) contacting said antibody- or antibodyfragment-expressing cells with a selected antigen; and (b) identifying ahost cell that binds to said selected antigen.
 17. The method of claim16, wherein said selected antigen is linked to a detectable label. 18.The method of claim 17, wherein said selected antigen is linked to a afluorescent label, a chemilluminescent label, a radioactive label,biotin, avidin, a magnetic bead or an enzyme that generates a coloredproduct upon contact with a chromogenic substrate.
 19. The method ofclaim 18, wherein said cells that bind to said selected antigen areidentified by a method comprising the steps of: (a) contacting saidplurality of cells with said detectably labeled antigen under conditionseffective to allow specific antigen-antibody binding; (b) removingnon-specifically bound antigen from said cells; and (c) identifying theantibody- or antibody fragment-expressing cells by detecting thepresence of the bound detectable label.
 20. The method of claim 19,wherein said cells that bind to said selected antigen are identified bya method comprising the steps of: (c) contacting said plurality of cellswith a fluorescently labeled antigen under conditions effective to allowspecific antigen-antibody binding; (d) subjecting said cells toautomated cell sorting; and (e) identifying the desired antibody orantibody fragment by detecting the fluorescently labeled sorted cells.21. The method of claim 20, wherein said cells are subjected to sortingby flow cytometry.
 22. The method of claim 20, wherein said cells aresubjected to a first and a second round of automated cell sorting. 23.The method of claim 22, wherein regrowth of sorted cells is conductedbetween said first and said second rounds of cell sorting.
 24. Themethod of claim 22, wherein said cells are subjected to a third and afourth round of automated cell sorting.
 25. The method of claim 18,wherein said selected antigen is linked to a magnetic bead.
 26. Themethod of claim 25, wherein cells that band said antigen are selectedare identified by a method comprising the steps of: (a) contacting saidplurality of cells with said magnetic bead labeled antigen underconditions effective to allow specific antigen-antibody binding; (b)subjecting said cells to magnetic sorting; and (c) identifying thedesired antibody- or antibody fragment by detecting the magentic beadlabeled sorted cells.
 27. The method of claim 4, wherein saidpolypeptide is an enzyme.
 28. The method of claim 27, wherein said cellsexpressing a desired enzyme are selected on the basis of enzymeactivity.
 29. The method of claim 28, wherein said enzyme activity issubstrate cleavage.
 30. The method of claim 29, wherein cleavage of saidsubstrate results in loss of quenching of a detectable signal.
 31. Themethod of claim 28, wherein said enzyme activity is substrate binding.32. The method of claim 31, wherein said binding results in thequenching of a detectable signal.
 33. The method of claim 31, whereinsaid binding results in the generation of a unique signal not found inthe absence of binding.
 34. The method of claim 28, wherein said enzymeactivity results in the association of a detectable signal with saidhost cell.
 35. The method of claim 34, further comprising sorting saidhost cell by flow cytometry.
 36. The method of claim 35, wherein saidcells are subjected to a second round of automated cell sorting.
 37. Themethod of claim 36, wherein regrowth of sorted cells is conductedbetween said first and said second rounds of cell sorting.
 38. A methodfor catalyzing a chemical reaction, comprising the steps of: (d)obtaining a host cell that expresses an enzyme or catalytic antibody onthe surface of the outer membrane; and (e) contacting said host cellwith a sample containing the necessary substrates for said chemicalreaction.
 39. The method of claim 38, wherein said host cell is a Gramnegative host cell.
 40. A method for stimulating an immune response,comprising administering to an animal a pharmaceutical compositioncomprising an immunologically effective amount of a host cell thatexpresses an antibody or antigen-combining antibody fragment on thesurface of the outer membrane.
 41. The method of claim 40, furthercomprising the step of obtaining from said animal an antibody.
 42. Themethod of claim 41, wherein said host cell is a Gram negative host cell.43. An isolated an purified antibody, or fragment thereof, that bindsimmunologically to digoxin, but does not bind immunologically todigitoxin.
 44. A single-chain antibody that binds immunologically todigoxin, but does not bind immunologically to digitoxin
 45. A host cellthat expresses, on its cell surface, a single-chain antibody that bindsimmunologically to a digoxin, but does not bind immunologically todigitoxin.